Extracellular expression of pectate lyase using Bacillus or Escherichia coli

ABSTRACT

The present invention relates to transformed bacterial hosts capable of expressing a pectate lyase enzyme endogenous to a strain of  Thermotoga maritima , especially a Bacillus or  E. coli  host cell, is useful in a method for producing the  Thermotoga maritima  pectate lyase. The  Thermotoga maritima  pectate lyase is useful for industrial use, e.g. for treatment of textiles.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority from orthe benefit of Danish application no. PA 2000 00621 filed Apr. 13, 2001,and U.S. provisional application No. 60/201,345, filed May 2, 2000 thecontents of which are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a transformed Bacillus or E.coli host capable of expressing an enzyme having pectate lyase activitywhich enzyme is endogenous to a strain of Thermotoga maritima, aBacillus or E. coli expression vector, a method for producing theThermotoga maritima pectate lyase in a Bacillus or E. coli host cell anda pectate lyase enzyme derived from a strain of Thermotoga maritime or asite-directed variant or mutant of this enzyme.

[0004] 2. Description of Related Art

[0005] Pectin polymers are important constituents of plant cell walls.Pectin is a hetero-polysaccharide with a backbone composed ofalternating homogalacturonan (smooth regions) and rhamnogalacturonan(hairy regions). The smooth regions are linear polymers of 1,4-linkedalpha-D-galacturonic acid. The galacturonic acid residues can bemethyl-esterified on the carboxyl group to a varying degree, usually ina non-random fashion with blocks of polygalacturonic acid beingcompletely methyl-esterified.

[0006] Pectinases can be classified according to their preferentialsubstrate, highly methyl-esterified pectin or low methyl-esterifiedpectin and polygalacturonic acid (pectate), and their reactionmechanism, beta-elimination or hydrolysis. Pectinases can be mainlyendo-acting, cutting the polymer at random sites within the chain togive a mixture of oligomers, or they may be exo-acting, attacking fromone end of the polymer and producing monomers or dimers. Severalpectinase activities acting on the smooth regions of pectin are includedin the classification of enzymes provided by the Enzyme Nomenclature(1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10),polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67),exo-polygalacturonate lyase (EC 4.2.2.9) andexo-poly-alpha-galacturonosidase (EC 3.2.1.82).

[0007] Pectate lyases have been cloned from different bacterial generasuch as Erwinia, Pseudomonas, Klebsiella and Xanthomonas. Cloning of apectate lyase has also been described from Bacillus subtilis (Nasser etal. (1993) FEBS 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994)Biosci. Biotech. Biochem. 58:947-949). Purification of pectate lyaseswith maximum activity in the pH range of 8-10 produced by Bacilluspumilus (Dave and Vaughn (1971) J. Bacteriol. 108:166-174), B. polymyxa(Nagel and Vaughn (1961) Arch. Biochem. Biophys. 93:344-352), B.stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol.26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci.31:838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J.Microbiol. 24:1164-1172) has been reported, however, no publication wasfound on cloning of pectate lyase encoding genes from these organisms.All the pectate lyases described require divalent cations for maximumactivity, calcium ions being the most stimulating.

[0008] The international patent application published as WO 99/27083discloses a pectate lyase cloned from Bacillus licheniformis. Theinternational patent application published as WO 99/27084 disclosespectate lyases cloned from Bacillus agaradhaerens, Bacillus haloduransand other Bacillus sp.

[0009] Nelson et al., Nature, 399:323-329 (1999): Evidence for lateralgene transfer between Archaea and Bacteria from genome sequence ofThermotoga maritima” disclose the complete genome sequence of Thermotogamaritima and the derived amino acid sequence. EMBL database entry(AE001722; SPTREMBL:Q9WYR4) suggests the product of the translated aminoacid sequence from section 34 of 136 of the complete Thermotoga maritimagenome to be a pectate lyase.

[0010] It is the object of the present invention to provide a pectatelyase enzyme having high performance in industrial processes, especiallyhigh temperature processes, a method for producing such a pectate lyasein high yields, preferably by means of a conventional fermentationtechnique involving extracellular production of the pectate lyase enzymeendogenous to a strain of Thermotoga maritima which technique makes theuse of pectate lyase in industrial applications economically feasible.

SUMMARY OF THE INVENTION

[0011] The inventors have now found that a species of Thermotogamaritima produces an enzyme having pectate lyase (EC 4.2.2.2) activityand have succeeded in cloning and expressing the pectate lyase enzyme ina Bacillus and an Escherichia coli host.

[0012] Accordingly, in a first aspect the present invention relates to aBacillus host transformed with a vector comprising a DNA sequence fromThermotoga maritima encoding for a pectate lyase polypeptide and capableof expressing the DNA sequence.

[0013] In a second aspect the present invention relates to a Bacillusexpression vector carrying an inserted DNA sequence from Thermotogamaritima encoding for a pectate lyase polypeptide.

[0014] In a third aspect, the invention relates to a method forproducing, in a Bacillus host cell, a polypeptide having pectate lyaseactivity, the method comprising the steps of

[0015] (a) growing under conditions to overproduce pectate lyasepolypeptide in a nutrient medium Bacillus host cells which have beentransformed with an expression cassette which includes, as operablyjoined components,

[0016] (i) a transcriptional and translational initiation regulatoryregion,

[0017] (ii) a DNA sequence encoding the pectate lyase polypeptide,

[0018] (iii) a transcriptional and translational termination regulatoryregion, wherein the regulatory regions are functional in the host, and

[0019] (iv) a selection marker gene for selecting transformed hostcells; and

[0020] (b) recovering the pectate lyase polypeptide.

[0021] Further, in its fourth aspect, the present invention relates toan enzyme having pectate lyase (EC 4.2.2.2) activity, which enzyme isendogenous to a strain of Thermotoga maritima or a variant of thisenzyme wherein one, two, three or four cysteine residues have beenaltered to other amino acid residues.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Microbial Sources

[0023] For the purpose of the present invention the term “obtained from”or “obtainable from” as used herein in connection with a specificsource, means that the enzyme is produced or can be produced by thespecific source, or by a cell in which a gene from the source have beeninserted.

[0024] It is at present contemplated that the pectate lyase of theinvention may be obtained from a strain of Thermotoga maritima.

[0025] In a preferred embodiment, the pectate lyase of the invention isobtained from the species Thermotoga maritima, DSM 3109, this strainbeing publicly available from Deutsche Sammiung von Mikroorganismen undZellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, FederalRepublic of Germany (DSM). It is at present contemplated that a DNAsequence encoding an enzyme with an amino acid sequence identity of atleast 85% to the enzyme of the invention may be obtained from otherstrains belonging to the species Thermotoga maritime.

[0026] In the present context, the term “enzyme preparation” is intendedto mean either be a conventional enzymatic fermentation product,possibly isolated and purified, from a single species of amicroorganism, such preparation usually comprising a number of differentenzymatic activities; or a mixture of monocomponent enzymes, preferablyenzymes derived from bacterial or fungal species by using conventionalrecombinant techniques, which enzymes have been fermented and possiblyisolated and purified separately and which may originate from differentspecies, preferably fungal or bacterial species; or the fermentationproduct of a microorganism which acts as a host cell for expression of arecombinant pectate lyase, but which microorganism simultaneouslyproduces other enzymes, e.g. xyloglucanases, proteases, or cellulases,being naturally occurring fermentation products of the microorganism,i.e. the enzyme complex conventionally produced by the correspondingnaturally occurring microorganism.

[0027] In the present context the term “expression vector” denotes a DNAmolecule, linear or circular, that comprises a segment encoding apolypeptide of interest operably linked to additional segments thatprovide for its transcription. Such additional segments may includepromoter and terminator sequences, and may optionally include one ormore origins of replication, one or more selectable markers, anenhancer, a polyadenylation signal, and the like. Expression vectors aregenerally derived from plasmid or viral DNA, or may contain elements ofboth. The expression vector of the invention may be any expressionvector that is conveniently subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which thevector is to be introduced. Thus, the vector may be an autonomouslyreplicating vector, i.e. a vector which exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g. a plasmid. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[0028] The term “recombinant expressed” or “recombinantly expressed”used herein in connection with expression of a polypeptide or protein isdefined according to the standard definition in the art. Recombinantexpression of a protein is generally performed by using an expressionvector as described immediately above.

[0029] The term “isolated”, when applied to a polynucleotide molecule,denotes that the polynucleotide has been removed from its naturalgenetic milieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985). The term “an isolatedpolynucleotide” may alternatively be termed “a cloned polynucleotide”.

[0030] When applied to a protein/polypeptide, the term “isolated”indicates that the protein is found in a condition other than its nativeenvironment. In a preferred form, the isolated protein is substantiallyfree of other proteins, particularly other homologous proteins (i.e.“homologous impurities” (see below)). It is preferred to provide theprotein in a greater than 40% pure form, more preferably greater than60% pure form.

[0031] Even more preferably it is preferred to provide the protein in ahighly purified form, i.e., greater than 80% pure, more preferablygreater than 95% pure, and even more preferably greater than 99% pure,as determined by SDS-PAGE.

[0032] The term “isolated protein/polypeptide may alternatively betermed “purified protein/polypeptide”.

[0033] The term “homologous impurities” means any impurity (e.g. anotherpolypeptide than the polypeptide of the invention) originating from thehomologous cell from which the polypeptide of the invention isoriginally obtained.

[0034] The term “obtained from” as used herein in connection with aspecific microbial source, means that the polynucleotide and/orpolypeptide produced by the specific source, or by a cell in which agene from the source have been inserted.

[0035] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator.

[0036] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules.

[0037] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0038] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0039] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0040] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0041] Polynucleotides

[0042] Within preferred embodiments of the invention an isolatedpolynucleotide of the invention will hybridize to similar sized regionsof SEQ ID NO:2, 4, 6 or 8, or a sequence complementary thereto, under atleast medium stringency conditions.

[0043] In particular polynucleotides of the invention will hybridize toa denatured double-stranded DNA probe comprising either the fullsequence shown in SEQ ID NO:2, 4, 6 or 8 or the sequence shown inpositions 88-1107 of SEQ ID NO:2, 4, 6 or 8 or any probe comprising asubsequence of SEQ ID NO:2, 4, 6 or 8 having a length of at least about100 base pairs under at least medium stringency conditions, butpreferably at high stringency conditions as described in detail below.Suitable experimental conditions for determining hybridization at mediumor high stringency between a nucleotide probe and a homologous DNA orRNA sequence involve presoaking of the filter containing the DNAfragments or RNA to hybridize in 5× SSC (Sodium chloride/Sodium citrate,Sambrook et al. 1989) for 10 min, and prehybridization of the filter ina solution of 5× SSC, 5× Denhardt's solution (Sambrook et al. 1989),0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrooket al. 1989), followed by hybridization in the same solution containinga concentration of 10 ng/ml of a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled(specific activity higher than 1×10⁹ cpm/pg) probe for 12 hours at ca.45° C. The filter is then washed twice for 30 minutes in 2× SSC, 0.5%SDS at least 60° C. (medium stringency), still more preferably at least65° C. (medium/high stringency), even more preferably at least 70° C.(high stringency), and even more preferably at least 75° C. (very highstringency).

[0044] Molecules to which the oligonucleotide probe hybridizes underthese conditions are detected using an X-ray film.

[0045] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. DNA and RNA encoding genes of interest can becloned in Gene Banks or DNA libraries by means of methods known in theart.

[0046] Polynucleotides encoding polypeptides having pectate lyaseactivity of the invention are then identified and isolated by, forexample, hybridization or PCR.

[0047] The present invention further provides counterpart polypeptidesand polynucleotides from different bacterial strains (orthologs orparalogs). Of particular interest are pectate lyase polypeptides fromstrains of Thermotoga maritima, examplified by the strain DSM 3109.

[0048] Species homologues of a polypeptide with pectate lyase activityof the invention can be cloned using information and compositionsprovided by the present invention in combination with conventionalcloning techniques. For example, a DNA sequence of the present inventioncan be cloned using chromosomal DNA obtained from a cell type thatexpresses the protein. Suitable sources of DNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from chromosomal DNA of a positivecell line. A DNA sequence of the invention encoding an polypeptidehaving pectate lyase activity can then be isolated by a variety ofmethods, such as by probing with probes designed from the sequencesdisclosed in the present specification and claims or with one or moresets of degenerate probes based on the disclosed sequences. A DNAsequence of the invention can also be cloned using the polymerase chainreaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the DNA library can be used to transform or transfect hostcells, and expression of the DNA of interest can be detected with anantibody (monoclonal or polyclonal) raised against the pectate lyasecloned from Thermotoga maritima, e.g. from DSM 3109, expressed andpurified as described in Materials and Methods and the examples, or byan activity test relating to a polypeptide having pectate lyaseactivity.

[0049] Polypeptides

[0050] The sequence of amino acids in positions 30-359 of SEQ ID NO: 3,5, 7 and 9, respectively, is a mature pectate lyase sequence comprisingthe catalytic active domain of the enzyme of the invention.

[0051] The present invention also provides pectate lyase polypeptidesthat are substantially homologous to the polypeptide of amino acids inposition 30-369 of SEQ ID NO:3 and species homologs (paralogs ororthologs) thereof. The term “substantially homologous” is used hereinto denote polypeptides having 85%, preferably at least 88%, morepreferably at least 90%, and even more preferably at least 95%, sequenceidentity to the sequence shown in amino acids nos. 30-369 of SEQ ID NO:3or its orthologs or paralogs. Such polypeptides will more preferably beat least 98% identical to the sequence shown in amino acids in positions30-369 of SEQ ID NO:3 or its orthologs or paralogs. Percent sequenceidentity is determined by conventional methods, by the Clustal method(Thompson, J. D., Higgins, D. G., and Gibson, T. J., (1994), NucleicAcids Research 22, 4673-4680) with the default settings of the Megalignprogram in the Lasergene package (DNAstart Inc., 1228 South Park Street,Madison, Wis. 53715). The settings for multiple alignment are: GAPpenalty of 10, and GAP length penalty 10; while the pair-wise alignmentparameters are GAP penalty of 3 and Ktuple of 1.

[0052] Sequence identity of polynucleotide molecules is determined bythe Clustal method (Thompson, J. D., Higgins, D. G., and Gibson, T. J.,(1994), Nucleic Acids Research 22, 4673-4680) with the default settingsof the Megalign program in the Lasergene package (DNAstart Inc., 1228South Park Street, Madison, Wis. 53715). The settings for multiplealignment are: GAP penalty of 10, and GAP length penalty 10; while thepair-wise alignment parameters are GAP penalty of 5 and Ktuple of 2.

[0053] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 2) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991. See, in general Ford et al., Protein Expressionand Purification 2: 95-107, 1991, which is incorporated herein byreference. DNAs encoding affinity tags are available from commercialsuppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; New EnglandBiolabs, Beverly, Mass.).

[0054] However, even though the changes described above preferably areof a minor nature, such changes may also be of a larger nature such asfusion of larger polypeptides of up to 300 amino acids or more both asamino- or carboxyl-terminal extensions to a polypeptide of the inventionhaving pectate lyase activity. TABLE 1 Conservative amino acidsubstitutions Basic: arginine lysine histidine Acidic: glutamic acidaspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

[0055] In addition to the 20 standard amino acids, non-standard aminoacids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyricacid, isovaline and a-methyl serine) may be substituted for amino acidresidues of a polypeptide according to the invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, or preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0056] Essential amino acids in the pectate lyase polypeptides of thepresent invention can be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (i.e pectate lyase activity) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Left. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with polypeptides relatedto a polypeptide according to the invention.

[0057] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination and/or shuffling followed bya relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988), Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989), WO95/17413, or WO95/22625. Briefly, these authors disclose methods for simultaneouslyrandomizing two or more positions in a polypeptide, orrecombination/shuffling of different mutations (WO95/17413, WO95/22625),followed by selecting for functional a polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0058] Mutagenesis/shuffling methods as disclosed above can be combinedwith high-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

[0059] Using the methods discussed above, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that aresubstantially homologous or identical to residues 30 to 369 of SEQ IDNO: 3 and retain the pectate lyase activity of the wild-type protein.

[0060] In a preferred embodiment, the present invention provides avariant enzyme of a pectate lyase endogeneous to Thermotoga maritima,the variant being a site directed variant with 3 removed cysteines, cf.Example 7 and 8. Preferably, the pectate lyase variant of the inventionhas amino acid substitutions in positions 161, 185 and 223 relative tothe amino acid numbering of SEQ ID NO: 3.

[0061] The pectate lyase enzyme of the invention may, in addition to theenzyme core comprising the catalytically domain, also comprise acellulose binding domain (CBD), the cellulose binding domain and enzymecore (the catalytically active domain) of the enzyme being operablylinked. The cellulose binding domain (CBD) may exist as an integral partthe encoded enzyme, or a CBD from another origin may be introduced intothe pectate lyase thus creating an enzyme hybrid. In this context, theterm “cellulose-binding domain” is intended to be understood as definedby Peter Tomme et al. “Cellulose-Binding Domains: Classification andProperties” in “Enzymatic Degradation of Insoluble Carbohydrates”, JohnN. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618,1996. This definition classifies more than 120 cellulose-binding domainsinto 10 families (I-X), and demonstrates that CBDs are found in variousenzymes such as cellulases, xylanases, mannanases, arabinofuranosidases,acetyl esterases and chitinases. CBDs have also been found in algae,e.g. the red alga Porphyra purpurea as a non-hydrolyticpolysaccharide-binding protein, see Tomme et al., op.cit. However, mostof the CBDs are from cellulases and xylanases, CBDs are found at the Nand C termini of proteins or are internal. Enzyme hybrids are known inthe art, see e.g. WO 90/00609 and WO 95/16782, and may be prepared bytransforming into a host cell a DNA construct comprising at least afragment of DNA encoding the cellulose-binding domain ligated, with orwithout a linker, to a DNA sequence encoding the pectate lyase andgrowing the host cell to express the fused gene. Enzyme hybrids may bedescribed by the following formula:

CBD-MR-X

[0062] wherein CBD is the N-terminal or the C-terminal region of anamino acid sequence corresponding to at least the cellulose-bindingdomain; MR is the middle region (the linker), and may be a bond, or ashort linking group preferably of from about 2 to about 100 carbonatoms, more preferably of from 2 to 40 carbon atoms; or is preferablyfrom about 2 to about 100 amino acids, more preferably of from 2 to 40amino acids; and X is an N-terminal or C-terminal region of apolypeptide encoded by the polynucleotide molecule of the invention.

[0063] Immunological Cross-Reactivity

[0064] Polyclonal antibodies, especially monospecific polyclonalantibodies, to be used in determining immunological cross-reactivity maybe prepared by use of a purified enzyme having pectate lyase activity.More specifically, antiserum against the pectate lyase of the inventionmay be raised by immunizing rabbits (or other rodents) according to theprocedure described by N. Axelsen et al. in: A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice,Blackwell Scientific Publications, 1982 (more specifically p. 27-31).Purified immunoglobulins may be obtained from the antisera, for exampleby salt precipitation ((NH₄)₂ SO₄), followed by dialysis and ionexchange chromatography, e.g. on DEAE-Sephadex. Immunochemicalcharacterization of proteins may be done either by Outcherlonydouble-diffusion analysis (O. Ouchterlony in: Handbook of ExperimentalImmunology (D. M. Weir, Ed.), Blackwell Scientific Publications, 1967,pp. 655-706), by crossed immunoelectrophoresis (N. Axelsen et al.,supra, Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsenet al., Chapter 2).

[0065] The Vector

[0066] As described in further detail below, the host of the inventionis transformed with a vector comprising a pectate lyase encoding DNAsequence. Preferably, the vector is integrated into the genome of thehost, more preferably it has been amplified on the genome.

[0067] In another preferred embodiment of the invention, the vector ispresent as an expression plasmid, preferably as a multi-copy plasmid.

[0068] The Bacillus expression vector of the invention carries aninserted pectate lyase-encoding DNA sequence. Preferably, the expressioncassette of the vector comprises regulatory regions from a Bacillus sp.,more preferably are such regulatory regions endogenous to the host.

[0069] In another aspect, the present invention further relates to amethod for optimisation of pectate lyase enzyme expression in a Bacillushost, the method comprising the steps of expression in the host of apectate lyase enzyme fused to a reporter molecule; and monitoring theconcentration of expressed enzyme in the supernatant of the fermentedhost by measuring the intrinsic property or properties of the reportermolecule.

[0070] In a preferred embodiment, the reporter molecule is a GreenFluorescent Protein, and the intrinsic property is fluorescenceemission.

[0071] In its fifth and sixth aspect, the invention relates to apolypeptide hybrid consisting essentially of a pectate lyase enzymefused to a green fluorescent protein, and to a method of producing sucha hybrid by expression in a Bacillus host, growth of the transformedhost under conditions whereby the transformed culture is substantiallyfree of untransformed cells; incubation of the transformed culture in anutrient medium, whereby the hybrid is overproduced; and recovery of thehybrid.

[0072] Expression of a Pectate Lyase Enzyme

[0073] Recombinant Expression Vectors

[0074] A recombinant vector comprising a DNA construct encoding theenzyme of the invention may be any vector conveniently subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. This introduction ofvector into the host cell is often referred to as the transformed hostcell. Such transformation indicates introduction of DNA into a host cellby using e.g. protoplasts, natural competent cells, transfection,conjugation, electroporation, or any equivalent method. Thus, the vectormay be an autonomously replicating vector, i.e. a vector existing as anextra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome in part or in its entirety and replicated together withthe chromosome(s) into which it has been integrated.

[0075] The vector is preferably an expression vector in which the DNAsequence encoding the pectate lyase enzyme of the invention is operablylinked to additional segments required for transcription of the DNA. Ingeneral, the expression vector is derived from plasmid or viral DNA, ormay contain elements of both. The term, “operably linked” indicates thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the CBD.

[0076] The promoter may be any DNA sequence showing transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

[0077] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilisalkaline protease gen, or the Bacillus pumilus xylosidase gene, or thephage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters. Alternatively, it is possible to design integration vectorssuch that the DNA encoding the pectate lyase enzyme will only becomefunctionally expressed once it is properly integrated into the hostgenome, e.g. downstream from a resident promoter.

[0078] The DNA sequence encoding the pectate lyase enzyme of theinvention may also, if necessary, be operably connected to a suitableterminator.

[0079] The recombinant vector of the invention may further comprise aDNA sequence enabling the vector to replicate in the host cell inquestion.

[0080] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g. antibiotics like kanamycin, chloramphenicol,erythromycin, tetracycline, spectinomycine, or the like, or resistanceto heavy metals or herbicides.

[0081] To direct a pectate lyase enzyme of the present invention intothe secretory pathway of the host cells, a secretory signal sequence(also known as a leader sequence, prepro sequence or pre sequence) maybe provided in the recombinant vector. The secretory signal sequence isjoined to the DNA sequence encoding the pectate lyase enzyme in thecorrect reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the. The secretory signalsequence may be that normally associated with the pectate lyase enzymeor may be from a gene encoding another secreted protein.

[0082] The procedures used to ligate the DNA sequences coding for thepresent pectate tyase enzyme, the promoter and optionally the terminatorand/or secretory signal sequence, respectively, or to assemble thesesequences by suitable PCR amplification schemes, and to insert them intosuitable vectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

[0083] Green Fluorescent Protein (GFP) has become a widely used reportermolecule for monitoring gene expression, tracers of cell lineage and asfusion tags for proteins (Crameri et al. (1996); Cubitt et al. (1995);International Patent Application PCT/DK96/00051).

[0084] GFP could be fused to pectate lyase enzymes creating a fusionprotein having the enzymatic property as well as the fluorescentproperties. The expression of this fusion protein could be used tomonitor the expressing of pectate lyase enzymes in Bacillus species andhereby be used to optimise expression levels of given pectate lyaseenzymes.

[0085] Host Cells

[0086] The cloned DNA molecule introduced into the host cell may beeither homologous or heterologous to the host in question. If homologousto the host cell, i.e. produced by the host cell in nature, it willtypically be operably connected to another promoter sequence or, ifapplicable, another secretory signal sequence and/or terminator sequencethan in its natural environment. The term “homologous” is intended toinclude a DNA sequence encoding an enzyme native to the host organism inquestion. The term “heterologous” is intended to include a DNA sequencenot expressed by the host cell in nature. Thus, the DNA sequence may befrom another organism, or it may be a synthetic sequence.

[0087] The host cell into which the cloned DNA molecule or therecombinant vector of the invention is introduced may be any cellcapable of producing the desired enzyme and includes bacteria, yeast,fungi and higher eukaryotic cells.

[0088] Examples of bacterial host cells which on cultivation are capableof producing the enzyme of the invention may be a gram-positive bacteriasuch as a strain of Bacillus, in particular Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus circulans, Bacillus coagulans,Bacillus megatherium, Bacillus stearothermophilus, Bacillus subtilis andBacillus thuringiensis, a strain of Lactobacillus, a strain ofStreptococcus, a strain of Streptomyces, in particular Streptomyceslividans and Streptomyces murinus, or the host cell may be agram-negative bacteria such as a strain of Escherichia coli.

[0089] The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. e.g. Sambrook et al., supra).

[0090] When expressing the enzyme in a bacteria such as Escherichiacoli, the enzyme may be retained in the cytoplasm, typically asinsoluble granules (known as inclusion bodies), or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed and the granules are recovered and denaturedafter which the enzyme is refolded by diluting the denaturing agent. Inthe latter case, the enzyme may be recovered from the periplasmic spaceby disrupting the cells, e.g. by sonication or osmotic shock, to releasethe contents of the periplasmic space and recovering the enzyme.

[0091] When expressing the enzyme in a gram-positive bacterium such as astrain of Bacillus or a strain of Streptomyces, the enzyme may beretained in the cytoplasm, or may be directed to the extra-cellularmedium by a bacterial secretion sequence.

[0092] Examples of a fungal host cell which on cultivation are capableof producing the enzyme of the invention is e.g. a strain of Aspergillusor Fusarium, in particular Aspergillus awamori, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, and Fusarium oxysporum, and astrain of Trichoderma, preferably Trichoderma harzianum, Trichodermareesei and Trichoderma viride.

[0093] Fungal cells may be transformed by a process involving protoplastformation and transformation of the protoplasts followed by regenerationof the cell wall in a manner known per se. The use of a strain ofAspergillus as a host cell is described in EP 238 023 (Novo NordiskA/S), the contents of which are hereby incorporated by reference.

[0094] Examples of a host cell of yeast origin which on cultivation arecapable of producing the enzyme of the invention is e.g. a strain ofHansenula sp., a strain of Kluyveromyces sp., in particularKluyveromyces lactis and Kluyveromyces marcianus, a strain of Pichiasp., a strain of Saccharomyces, in particular Saccharomycescarlsbergensis, Saccharomyces cerevisae, Saccharomyces kluyveri andSaccharomyces uvarum, a strain of Schizosaccharomyces sp., in particularSchizosaccharomyces pombe, and a strain of Yarrowia sp., in particularYarrowia lipolytica.

[0095] Examples of a host cell of plant origin which on cultivation arecapable of producing the enzyme of the invention is e.g. a plant cell ofSolanum tuberosum or Nicotiana tabacum.

[0096] Method of Producing a Pectate Lyase Enzyme

[0097] In another aspect, the present invention also relates to a methodof producing the enzyme preparation of the invention, the methodcomprising culturing a microorganism capable of producing the pectatelyase under conditions permitting the production of the enzyme, andrecovering the enzyme from the culture. Culturing may be carried outusing conventional fermentation techniques, e.g. culturing in shakeflasks or fermentors with agitation to ensure sufficient aeration on agrowth medium inducing production of the pectate lyase enzyme. Thegrowth medium may contain a conventional N-source such as peptone, yeastextract or casamino acids, a reduced amount of a conventional C-sourcesuch as dextrose or sucrose, and an inducer such as pectinase orcomposit plant substrates such as cereal brans (e.g. wheat bran or ricehusk). The recovery may be carried out using conventional techniques,e.g. separation of bio-mass and supernatant by centrifugation orfiltration, recovery of the supernatant or disruption of cells if theenzyme of interest is intracellular, perhaps followed by furtherpurification as described in EP 0 406 314 or by crystallization asdescribed in WO 97/15660.

[0098] Further, the present invention provides a method of producing anisolated enzyme according to the invention, wherein a suitable hostcell, which has been transformed with a DNA sequence encoding theenzyme, is cultured under conditions permitting the production of theenzyme, and the resulting enzyme is recovered from the culture.

[0099] As defined herein, an isolated polypeptide (e.g. an enzyme) is apolypeptide which is essentially free of other polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

[0100] The term “isolated polypeptide” may alternatively be termed“purified polypeptide”.

[0101] When an expression vector comprising a DNA sequence encoding theenzyme is transformed into a heterologous host cell it is possible toenable heterologous recombinant production of the enzyme of theinvention.

[0102] Thereby it is possible to make a highly purified or monocomponentpectate lyase composition, characterized in being free from homologousimpurities.

[0103] In this context, homologous impurities mean any impurities (e.g.other polypeptides than the enzyme of the invention) which originatefrom the homologous cell where the enzyme of the invention is originallyobtained from.

[0104] In the present invention the homologous host cell may be a strainof Thermotoga maritima.

[0105] The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed pectate lyase enzyme may conveniently be secreted into theculture medium and may be recovered therefrom by well-known proceduresincluding separating the cells from the medium by centrifugation orfiltration, precipitating proteinaceous components of the medium bymeans of a salt such as ammonium sulfate, followed by chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

[0106] The present invention also relates to a transgenic plant, plantpart or plant cell which has been transformed with a DNA sequenceencoding the pectate lyase of the invention so as to express and producethis enzyme in recoverable quantities. The enzyme may be recovered fromthe plant or plant part.

[0107] The transgenic plant can be dicotyledonous or monocotyledonous,for short a dicot or a monocot. Examples of monocot plants are grasses,such as meadow grass (blue grass, Poa), forage grass such as festuca,lolium, temperate grass, such as Agrostis, and cereals, e.g. wheat,oats, rye, barley, rice, sorghum and maize (corn).

[0108] Examples of dicot plants are tobacco, legumes, such as lupins,potato, sugar beet, pea, bean and soybean, and cruciferous (familyBrassicaceae), such as cauliflower, oil seed rape and the closelyrelated model organism Arabidopsis thaliana.

[0109] Examples of plant parts are stem, callus, leaves, root, fruits,seeds, and tubers. In the present context, also specific plant tissues,such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes andcytoplasm are considered to be a plant part. Furthermore, any plantcell, whatever the tissue origin, is considered to be a plant part.

[0110] Also included within the scope of the invention are the progenyof such plants, plant parts and plant cells.

[0111] The transgenic plant or plant cell expressing the enzyme of theinvention may be constructed in accordance with methods known in theart. In short the plant or plant cell is constructed by incorporatingone or more expression constructs encoding the enzyme of the inventioninto the plant host genome and propagating the resulting modified plantor plant cell into a transgenic plant or plant cell.

[0112] Conveniently, the expression construct is a DNA construct whichcomprises a gene encoding the enzyme of the invention in operableassociation with appropriate regulatory sequences required forexpression of the gene in the plant or plant part of choice.Furthermore, the expression construct may comprise a selectable markeruseful for identifying host cells into which the expression constructhas been integrated and DNA sequences necessary for introduction of theconstruct into the plant in question (the latter depends on the DNAintroduction method to be used).

[0113] The choice of regulatory sequences, such as promoter andterminator sequences and optionally signal or transit sequences isdetermined, e.g. based on when, where and how the enzyme is desired tobe expressed. For instance, the expression of the gene encoding theenzyme of the invention may be constitutive or inducible, or may bedevelopmental, stage or tissue specific, and the gene product may betargeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are e.g. described by Tague et al, Plant, Phys.,86, 506, 1988.

[0114] For constitutive expression the 35S-CaMV promoter may be used(Franck et al., 1980. Cell 21: 285-294). Organ-specific promoters may egbe a promoter from storage sink tissues such as seeds, potato tubers,and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), orfrom metabolic sink tissues such as meristems (Ito et al., 1994. PlantMol. Biol. 24: 863-878), a seed specific promoter such as the glutelin,prolamin, globulin or albumin promoter from rice (Wu et al., Plant andCell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia fabapromoter from the legumin B4 and the s unknown seed protein gene fromVicia faba described by Conrad U. et al, Journal of Plant PhysiologyVol. 152, No. 6 pp. 708-711 (1998), a promotter from a seed oil bodyprotein (Chen et al., Plant and cell physiology vol. 39, No. 9 pp.935-941 (1998), the storage protein napA promoter from Brassica napus,or any other seed specific promoter known in the art, e.g. as describedin WO 91/14772. Furthermore, the promoter may be a leaf specificpromoter such as the rbcs promoter from rice or tomato (Kyozuka et al.,Plant Physiology Vol. 102, No. 3 pp. 991-1000 (1993), the chlorellavirus adenine methyltransferase gene promoter (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol. 26, No. 1 pp. 85-93 (1994), or the aldPgene promoter from rice (Kagaya et al., Molecular and General GeneticsVol. 248, No. 6 pp. 668-674 (1995), or a wound inducible promoter suchas the potato pin2 promoter (Xu et al, Plant Molecular Biology Vol. 22,No. 4 pp. 573-588 (1993).

[0115] A promoter enhancer element may be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron placed between the promoter and thenucleotide sequence encoding the enzyme. For instance, Xu et al. op citdisclose the use of the first intron of the rice actin 1 gene to enhanceexpression.

[0116] The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

[0117] The DNA construct is incorporated into the plant genome accordingto conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,micro injection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn.8, 535, 1990; Shimamoto et al, Nature, 338, 274, 1989).

[0118] Presently, Agrobacterium tumefaciens mediated gene transfer isthe method of choice for generating transgenic dicots (for reviewHooykas & Schilperoort, 1992. Plant Mol. Biol. 19: 15-38), however itcan also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992. Plant J. 2: 275-281; Shimamoto, 1994. Curr. Opin.Biotechnol. 5: 158-162; Vasil et al., 1992. Bio/Technology 10: 667-674).An alternative method for transformation of monocots is based onprotoplast transformation as described by Omirulleh S, et al., PlantMolecular biology Vol. 21, No. 3 pp. 415-428 (1993).

[0119] Following transformation, the transformants having incorporatedthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

[0120] Enzyme Compositions

[0121] In a still further aspect, the present invention relates to anenzyme composition comprising an enzyme exhibiting pectate lyaseactivity as described above.

[0122] The enzyme composition of the invention may, in addition to thepectate lyase of the invention, comprise one or more other enzyme types,for instance hemicellulase such as xylanase and mannanase, cellulase orendo-β-1,4-glucanase components, chitinase, lipase, esterase, pectinase,xyloglucanase, cutinase, phytase, oxidoreductase (peroxidase,haloperoxidase, oxidase, laccase), protease, amylase, reductase,phenoloxidase, ligninase, pullulanase, pectate lyase, pectin acetylesterase, polygalacturonase, rhamnogalacturonase, pectin lyase, pectinmethylesterase, cellobiohydrolase, transglutaminase; or mixturesthereof.

[0123] The enzyme composition may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the enzyme composition may be in the form ofa granulate or a microgranulate. The enzyme to be included in thecomposition may be stabilized in accordance with methods known in theart.

[0124] Uses

[0125] Pectate lyases have potential uses in a lot of differentindustries and applications. Examples are given below of preferred usesof the enzyme composition of the invention. The dosage of the enzymecomposition of the invention and other conditions under which thecomposition is used may be determined based on methods known in the art.

[0126] It is contemplated that the pectate lyase of the invention isuseful as an ingredient of a laundry detergent composition or fortreatment of textile fabric, especially for scouring of textile ortextile fabric at elevated temperatures.

[0127] Use in the Detergent Industry

[0128] During washing and wearing, dyestuff from dyed fabrics or garmentwill conventionally bleed from the fabric, which then looks faded andworn. Removal of surface fibers from the fabric will partly restore theoriginal colours and looks of the fabric. By the term “colourclarification”, as used herein, is meant the partly restoration of theinitial colours of fabric or garment throughout multiple washing cycles.

[0129] The term “de-pilling” denotes removing of pills from the fabricsurface.

[0130] The term “soaking liquor” denotes aqueous liquor in which laundrymay be immersed prior to being subjected to a conventional washingprocess. The soaking liquor may contain one or more ingredientsconventionally used in a washing or laundering process.

[0131] The term “washing liquor” denotes aqueous liquor in which laundryis subjected to a washing process, i.e. usually a combined chemical andmechanical action either manually or in a washing machine.Conventionally, the washing liquor is an aqueous solution of a powder orliquid detergent composition.

[0132] The term “rinsing liquor” denotes aqueous liquor in which laundryis immersed and treated, conventionally immediately after beingsubjected to a washing process, in order to rinse the laundry, i.e.essentially remove the detergent solution from the laundry. The rinsingliquor may contain a fabric conditioning or softening composition.

[0133] The laundry subjected to the method of the present invention maybe conventional washable laundry. Preferably, the major part of thelaundry is sewn or unsown fabrics, including knits, wovens, denims,yarns, and towelling, made from cotton, cotton blends or natural ormanmade cellulosics (e.g. originating from xylan-containing cellulosefibers such as from wood pulp) or blends thereof. Examples of blends areblends of cotton or rayon/viscose with one or more companion materialsuch as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, flax/linen, jute, cellulose acetate fibers, lyocell).

[0134] Detergent Disclosure and Examples

[0135] Surfactant System

[0136] The detergent compositions according to the present inventioncomprise a surfactant system, wherein the surfactant can be selectedfrom nonionic and/or anionic and/or cationic and/or ampholytic and/orzwifterionic and/or semi-polar surfactants.

[0137] The surfactant is typically present at a level from 0.1% to 60%by weight.

[0138] The surfactant is preferably formulated to be compatible withenzyme components present in the composition. In liquid or gelcompositions the surfactant is most preferably formulated in such a waythat it promotes, or at least does not degrade, the stability of anyenzyme in these compositions.

[0139] Preferred systems to be used according to the present inventioncomprise as a surfactant one or more of the nonionic and/or anionicsurfactants described herein, preferably sodium alkylether sulphate(AExS).

[0140] Polyethylene, polypropylene, and polybutylene oxide conden-satesof alkyl phenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being pre-ferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and TritonX-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

[0141] The condensation products of primary and secondary aliphaticalcohols with about 1 to about 25 moles of ethylene oxide are suitablefor use as the nonionic surfactant of the nonionic surfactant systems ofthe present invention. The alkyl chain of the aliphatic alcohol caneither be straight or branched, primary or secondary, and generallycontains from about 8 to about 22 carbon atoms. Preferred are thecondensation products of alcohols having an alkyl group containing fromabout 8 to about 20 carbon atoms, more preferably from about 10 to about18 carbon atoms, with from about 2 to about 10 moles of ethylene oxideper mole of alcohol. About 2 to about 7 moles of ethylene oxide and mostpreferably from 2 to 5 moles of ethylene oxide per mole of alcohol arepresent in said condensation products. Examples of commerciallyavailable nonionic surfactants of this type include Tergitol™ 15-S-9(The condensation product of C₁₁-C₁₅ linear alcohol with 9 molesethylene oxide), Tergitol™ 24-L-6 NMW (the condensation product ofC₁₂-C₁₄ primary alcohol with 6 moles ethylene oxide with a narrowmolecular weight distribution), both marketed by Union CarbideCorporation; Neodol™ 45-9 (the condensation product of C₁₄-C₁₅ linearalcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensationproduct of C₁₂-C₁₃ linear alcohol with 3.0 moles of ethylene oxide),Neodol™ 45-7 (the condensation product Of C₁₄-C₁₅ linear alcohol with 7moles of ethylene oxide), Neodol™ 45-5 (the condensation product ofC₁₄-C₁₅ linear alcohol with 5 moles of ethylene oxide) marketed by ShellChemical Company, Kyro™ EOB (the condensation product of C₁₃-C₁₅ alcoholwith 9 moles ethylene oxide), marketed by The Procter & Gamble Company,and Genapol LA 050 (the condensation product of C₁₂-C₁₄ alcohol with 5moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB inthese products is from 8-11 and most preferred from 8-10.

[0142] Also useful as the nonionic surfactant of the surfactant systemsof the present invention are alkylpolysaccharides disclosed in U.S. Pat.No. 4,565,647, having a hydrophobic group containing from about 6 toabout 30 carbon atoms, preferably from about 10 to about 16 carbon atomsand a polysaccharide, e.g. a polyglycoside, hydrophilic group containingfrom about 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

[0143] The preferred alkylpolyglycosides have the formula

R²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)

[0144] wherein R² is selected from the group consisting of alkyl,alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof inwhich the alkyl groups contain from about 10 to about 18, preferablyfrom about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t isfrom 0 to about 10, preferably 0; and x is from about 1.3 to about 10,preferably from about 1.3 to about 3, most preferably from about 1.3 toabout 2.7. The glycosyl is preferably derived from glucose. To preparethese compounds, the alcohol or alkylpolyethoxy alcohol is formed firstand then reacted with glucose, or a source of glucose, to form theglucoside (attachment at the 1-position). The additional glycosyl unitscan then be attached between their 1-position and the preceding glycosylunits 2-, 3-, 4-, and/or 6-position, preferably predominantly the2-position.

[0145] The condensation products of ethylene oxide with a hydrophobicbase formed by the condensation of propylene oxide with propylene glycolare also suitable for use as the additional nonionic surfactant systemsof the present invention. The hydrophobic portion of these compoundswill preferably have a molecular weight from about 1500 to about 1800and will exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

[0146] Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

[0147] Preferred for use as the nonionic surfactant of the surfactantsystems of the present invention are polyethylene oxide condensates ofalkyl phenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₁₈alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

[0148] Highly preferred nonionic surfactants are polyhydroxy fatty acidamide surfactants of the formula

[0149] wherein R¹ is H, or R¹ is C₁₋₄ hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₆₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

[0150] Highly preferred anionic surfactants include alkyl alkoxylatedsulfate surfactants. Examples hereof are water soluble salts or acids ofthe formula RO(A)_(m)SO3M wherein R is an unsubstituted C₁₀-C-₂₄ alkylor hydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably aC₁₂-C₂₀ alkyl or hydroxyalkyl, more preferably C₁₂-C₁₈ alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperdinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

[0151] Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52 (1975), pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

[0152] The preferred alkyl ester sulfonate surfactant, especially forlaundry applications, comprise alkyl ester sulfonate surfactants of thestructural formula:

[0153] wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, orcombination thereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, orcombination thereof, and M is a cation which forms a water soluble saltwith the alkyl ester sulfonate. Suitable salt-forming cations includemetals such as sodium, potassium, and lithium, and substituted orunsubstituted ammonium cations, such as monoethanolamine,diethonolamine, and triethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl,and R⁴ is methyl, ethyl or isopropyl. Especially preferred are themethyl ester sulfonates wherein R³ is C₁₀-C₁₆ alkyl.

[0154] Other suitable anionic surfactants include the alkyl sulfatesurfactants which are water soluble salts or acids of the formula ROSO₃Mwherein R preferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl orhydroxyalkyl having a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metalcation (e.g. sodium, potassium, lithium), or ammonium or substitutedammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations andquaternary ammonium cations such as tetramethyl-ammonium and dimethylpiperdinium cations and quaternary ammonium cations derived fromalkylamines such as ethylamine, diethylamine, triethylamine, andmixtures thereof, and the like). Typically, alkyl chains of C₁₂-C₁₆ arepreferred for lower wash temperatures (e.g. below about 50° C.) andC₁₆-C₁₈ alkyl chains are preferred for higher wash temperatures (e.g.above about 50° C.).

[0155] Other anionic surfactants useful for detersive purposes can alsobe included in the laundry detergent compositions of the presentinvention. Theses can include salts (including, for example, sodium,potassium, ammonium, and substituted ammonium salts such as mono-di- andtriethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C₆-C₁₂ diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH₂CH₂O)_(k)—CH₂COO-M+wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is asoluble salt forming cation. Resin acids and hydrogenated resin acidsare also suitable, such as rosin, hydrogenated rosin, and resin acidsand hydrogenated resin acids present in or derived from tall oil.

[0156] Alkylbenzene sulfonates are highly preferred. Especiallypreferred are linear (straight-chain) alkyl benzene sulfonates (LAS)wherein the alkyl group preferably contains from 10 to 18 carbon atoms.

[0157] Further examples are described in “Surface Active Agents andDetergents” (Vol. I and II by Schwartz, Perrry and Berch). A variety ofsuch surfactants are also generally disclosed in U.S. Pat. No.3,929,678, (Column 23, line 58 through Column 29, line 23, hereinincorporated by reference).

[0158] When included therein, the laundry detergent compositions of thepresent invention typically comprise from about 1% to about 40%,preferably from about 3% to about 20% by weight of such anionicsurfactants.

[0159] The laundry detergent compositions of the present invention mayalso contain cationic, ampholytic, zwifterionic, and semi-polarsurfactants, as well as the nonionic and/or anionic surfactants otherthan those already described herein.

[0160] Cationic detersive surfactants suitable for use in the laundrydetergent compositions of the present invention are those having onelong-chain hydrocarbyl group. Examples of such cationic surfactantsinclude the ammonium surfactants such as alkyltrimethylammoniumhalogenides, and those surfactants having the formula:

[R²(OR³)_(y)] [R⁴(OR³)_(y)]₂R⁵N+X—

[0161] wherein R² is an alkyl or alkyl benzyl group having from about 8to about 18 carbon atoms in the alkyl chain, each R³ is selected formthe group consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—,—CH₂CH₂CH₂—, and mixtures thereof; each R⁴ is selected from the groupconsisting of C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structuresformed by joining the two R groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, whereinR⁶ is any hexose or hexose polymer having a molecular weight less thanabout 1000, and hydrogen when y is not 0; R⁵ is the same as R⁴ or is analkyl chain, wherein the total number of carbon atoms or R²plus R⁵ isnot more than about 18; each y is from 0 to about 10, and the sum of they values is from 0 to about 15; and X is any compatible anion.

[0162] Highly preferred cationic surfactants are the water solublequaternary ammonium compounds useful in the present composition havingthe formula:

R₁R₂R₃R₄N⁺X⁻C  (i)

[0163] wherein R₁ is C₈-C₁₆ alkyl, each of R₂, R₃ and R₄ isindependently C₁-C₄ alkyl, C₁-C₄ hydroxy alkyl, benzyl, and—(C₂H₄₀)_(x)H where x has a value from 2 to 5, and X is an anion. Notmore than one of R₂, R₃ or R₄ should be benzyl.

[0164] The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularlywhere the alkyl group is a mixture of chain lengths derived from coconutor palm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

[0165] Preferred groups for R₂R₃ and R₄ are methyl and hydroxyethylgroups and the anion X may be selected from halide, methosulphate,acetate and phosphate ions.

[0166] Examples of suitable quaternary ammonium compounds of formulae(i) for use herein are:

[0167] coconut trimethyl ammonium chloride or bromide;

[0168] coconut methyl dihydroxyethyl ammonium chloride or bromide;

[0169] decyl triethyl ammonium chloride;

[0170] decyl dimethyl hydroxyethyl ammonium chloride or bromide;

[0171] C₁₂-₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;

[0172] coconut dimethyl hydroxyethyl ammonium chloride or bromide;

[0173] myristyl trimethyl ammonium methyl sulphate;

[0174] lauryl dimethyl benzyl ammonium chloride or bromide;

[0175] lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;

[0176] choline esters (compounds of formula (i) wherein R₁ is

[0177] alkyl and R₂R₃R₄ are methyl).

[0178] di-alkyl imidazolines [compounds of formula (i)].

[0179] Other cationic surfactants useful herein are also described inU.S. Pat. No. 4,228,044 and in EP 000 224.

[0180] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 25%, preferablyfrom about 1% to about 8% by weight of such cationic surfactants.

[0181] Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

[0182] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such ampholytic surfactants.

[0183] Zwitterionic surfactants are also suitable for use in laundrydetergent compositions. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds. SeeU.S. Pat. No. 3,929,678 (column 19, line 38 through column 22, line 48)for examples of zwitterionic surfactants.

[0184] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such zwitterionic surfactants.

[0185] Semi-polar nonionic surfactants are a special category ofnonionic surfactants which include water-soluble amine oxides containingone alkyl moiety of from about 10 to about 18 carbon atoms and 2moieties selected from the group consisting of alkyl groups andhydroxyalkyl groups containing from about 1 to about 3 carbon atoms;watersoluble phosphine oxides containing one alkyl moiety of from about10 to about 18 carbon atoms and 2 moieties selected from the groupconsisting of alkyl groups and hydroxyalkyl groups containing from about1 to about 3 carbon atoms; and water-soluble sulfoxides containing onealkyl moiety from about 10 to about 18 carbon atoms and a moietyselected from the group consisting of alkyl and hydroxyalkyl moieties offrom about 1 to about 3 carbon atoms.

[0186] Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

[0187] wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group ormixtures thereof containing from about 8 to about 22 carbon atoms; R⁴ isan alkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

[0188] These amine oxide surfactants in particular include C₁₀-C₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

[0189] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such semi-polar nonionicsurfactants.

[0190] Builder System

[0191] The compositions according to the present invention may furthercomprise a builder system. Any conventional builder system is suitablefor use herein including aluminosilicate materials, silicates,polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetate, metal ion sequestrants such as aminopolyphosphonates,particularly ethylenediamine tetramethylene phosphonic acid anddiethylene triamine pentamethylenephosphonic acid. Though less preferredfor obvious environmental reasons, phosphate builders can also be usedherein.

[0192] Suitable builders can be an inorganic ion exchange material,commonly an inorganic hydrated aluminosilicate material, moreparticularly a hydrated synthetic zeolite such as hydrated zeolite A, X,B, HS or MAP.

[0193] Another suitable inorganic builder material is layered silicate,e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consistingof sodium silicate (Na₂Si₂O₅).

[0194] Suitable polycarboxylates containing one carboxy group includelactic acid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

[0195] Polycarboxylates containing four carboxy groups includeoxydisuccinates disclosed in British Patent No. 1,261,829,1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane tetrac7arboxylatescontaining sulfo substituents include the sulfosuccinate derivativesdisclosed in British Patent Nos. 1,398,421 and 1,398,422 and in U.S.Pat. No. 3,936,448, and the sulfonated pyrolysed citrates described inBritish Patent No. 1,082,179, while polycarboxylates containingphosphone substituents are disclosed in British Patent No. 1,439,000.

[0196] Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates, 2,3,4,5-tetrahydro-furan-cis, cis,cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates,2,2,5,5,-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

[0197] Of the above, the preferred polycarboxylates arehydroxy-carboxylates containing up to three carboxy groups per molecule,more particularly citrates.

[0198] Preferred builder systems for use in the present compositionsinclude a mixture of a water-insoluble aluminosilicate builder such aszeolite A or of a layered silicate (SKS-6), and a water-solublecarboxylate chelating agent such as citric acid.

[0199] A suitable chelant for inclusion in the detergent compositions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

[0200] Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

[0201] Other builder materials that can form part of the builder systemfor use in granular compositions include inorganic materials such asalkali metal carbonates, bicarbonates, silicates, and organic materialssuch as the organic phosphonates, amino polyalkylene phosphonates andamino polycarboxylates.

[0202] Other suitable water-soluble organic salts are the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated form each other bynot more than two carbon atoms.

[0203] Polymers of this type are disclosed in GB-A-1,596,756. Examplesof such salts are polyacrylates of MW 2000-5000 and their copolymerswith maleic anhydride, such copolymers having a molecular weight of from20,000 to 70,000, especially about 40,000.

[0204] Detergency builder salts are normally included in amounts of from5% to 80% by weight of the composition. Preferred levels of builder forliquid detergents are from 5% to 30%.

[0205] Enzymes

[0206] Preferred detergent compositions, in addition to the enzymepreparation of the invention, comprise other enzyme(s) which providescleaning performance and/or fabric care benefits.

[0207] Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g. laccases).

[0208] Proteases: Any protease suitable for use in alkaline solutionscan be used. Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically orgenetically modified mutants are included. The protease may be a serineprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus, e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describedin WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. ofporcine or bovine origin) and the Fusarium protease described in WO89/06270.

[0209] Preferred commercially available protease enzymes include thosesold under the trade names Alcalase, Savinase, Primase, Durazym, andEsperase by Novo Nordisk A/S (Denmark), those sold under the tradenameMaxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP byGenencor International, and those sold under the tradename Opticlean andOptimase by Solvay Enzymes. Protease enzymes may be incorporated intothe compositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

[0210] Lipases: Any lipase suitable for use in alkaline solutions can beused. Suitable lipases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included.

[0211] Examples of useful lipases include a Humicola lanuginosa lipase,e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor mieheilipase, e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

[0212] Furthermore, a number of cloned lipases may be useful, includingthe Penicillium camembertii lipase described by Yamaguchi et al.,(1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. etal., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipasessuch as a R. delemar lipase (Hass, M. J et al., (1991), Gene109,117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci.Biotech. Biochem. 56, 716-719) and a R. oryzae lipase.

[0213] Other types of lipolytic enzymes such as cutinases may also beuseful, e.g., a cutinase derived from Pseudomonas mendocina as describedin WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g.described in WO 90/09446).

[0214] Especially suitable lipases are lipases such as M1 Lipase™, Lumafast™ and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (NovoNordisk A/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

[0215] The lipases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0216] Amylases: Any amylase (a and/or b) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, a-amylases obtained from a special strainof B. licheniformis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™(available from Genencor).

[0217] The amylases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0218] Cellulases: Any cellulase suitable for use in alkaline solutionscan be used. Suitable cellulases include those of bacterial or fungalorigin. Chemically or genetically modified mutants are included.Suitable cellulases are disclosed in U.S. Pat. No. 4,435,307 whichdiscloses fungal cellulases produced from Humicola insolens, in WO96/34108 and WO 96/34092 which disclose bacterial alkalophiliccellulases (BCE 103) from Bacillus, and in WO 94/21801, U.S. Pat. No.5,475,101 and U.S. Pat. No. 5,419,778 which disclose EG III cellulasesfrom is Trichoderma. Especially suitable cellulases are the cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in European patent application No. 0 495 257. Commerciallyavailable cellulases include Celluzyme™ and Carezyme™ produced by astrain of Humicola insolens (Novo Nordisk A/S), KAC-500(B)™ (KaoCorporation), and Puradax™ (Genencor International).

[0219] Cellulases are normally incorporated in the detergent compositionat a level of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

[0220] Peroxidases/Oxidases: Peroxidase enzymes are used in combinationwith hydrogen peroxide or a source thereof (e.g. a percarbonate,perborate or persulfate). Oxidase enzymes are used in combination withoxygen. Both types of enzymes are used for “solution bleaching”, i.e. toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in e.g. WO 94112621 and WO95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

[0221] Peroxidase and/or oxidase enzymes are normally incorporated inthe detergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

[0222] Mixtures of the above mentioned enzymes are encompassed herein,in particular a mixture of a protease, an amylase, a lipase and/or acellulase.

[0223] The enzyme of the invention, or any other enzyme incorporated inthe detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

[0224] Bleaching Agents

[0225] Additional optional detergent ingredients that can be included inthe detergent compositions of the present invention include bleachingagents such as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present oxygen bleaching compounds willtypically be present at levels of from about 1% to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g. granular detergents.

[0226] The bleaching agent component for use herein can be any of thebleaching agents useful for detergent compositions including oxygenbleaches as well as others known in the art.

[0227] The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

[0228] One category of oxygen bleaching agent that can be usedencompasses percarboxylic acid bleaching agents and salts thereof.Suitable examples of this class of agents include magnesiummonoperoxyphthalate hexahydrate, the magnesium salt of meta-chloroperbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid anddiperoxydodecanedioic acid. Such bleaching agents are disclosed in U.S.Pat. No. 4,483,781, U.S. Pat. No. 740,446, EP 0 133 354 and U.S. Pat.No. 4,412,934. Highly preferred bleaching agents also include6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No.4,634,551.

[0229] Another category of bleaching agents that can be used encompassesthe halogen bleaching agents. Examples of hypohalite bleaching agents,for example, include trichloro isocyanuric acid and the sodium andpotassium dichloroisocyanurates and N-chloro and N-bromo alkanesulphonamides. Such materials are normally added at 0.5-10% by weight ofthe finished product, preferably 1-5% by weight.

[0230] The hydrogen peroxide releasing agents can be used in combinationwith bleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8(6-octanamido-caproyl) oxybenzene-sulfonate,C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

[0231] Useful bleaching agents, including peroxyacids and bleachingsystems comprising bleach activators and peroxygen bleaching compoundsfor use in cleaning compositions according to the invention aredescribed in application U.S. Ser. No. 08/136,626.

[0232] The hydrogen peroxide may also be present by adding an enzymaticsystem (i.e. an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

[0233] Bleaching agents other than oxygen bleaching agents are alsoknown in the art and can be utilized herein. One type of non-oxygenbleaching agent of particular interest includes photoactivated bleachingagents such as the sulfonated zinc and/or aluminium phthalocyanines.These materials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

[0234] Bleaching agents may also comprise a manganese catalyst. Themanganese catalyst may, e.g., be one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching”, Nature369, 1994, pp. 637-639.

[0235] Suds Suppressors

[0236] Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwater-dispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

[0237] A preferred silicone suds controlling agent is disclosed in U.S.Pat. No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol whichare commercially available under the trade name Isofol 12 R.

[0238] Such suds suppressor system are described in European PatentApplication EP 0 593 841.

[0239] Especially preferred silicone suds controlling agents aredescribed in European Patent Application No. 92201649.8. Saidcompositions can comprise a silicone/silica mixture in combination withfumed nonporous silica such as Aerosil^(R).

[0240] The suds suppressors described above are normally employed atlevels of from 0.001% to 2% by weight of the composition, preferablyfrom 0.01% to 1% by weight.

[0241] Other Components

[0242] Other components used in detergent compositions may be employedsuch as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

[0243] Especially suitable encapsulating materials are water solublecapsules which consist of a matrix of polysaccharide and polyhydroxycompounds such as described in GB 1,464,616.

[0244] Other suitable water soluble encapsulating materials comprisedextrins derived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

[0245] Antiredeposition and soil suspension agents suitable hereininclude cellulose derivatives such as methylcellulose,carboxymethylcellulose and hydroxyethylcellulose, and homo- orco-polymeric polycarboxylic acids or their salts. Polymers of this typeinclude the polyacrylates and maleic anhydride-acrylic acid copolymerspreviously mentioned as builders, as well as copolymers of maleicanhydride with ethylene, methylvinyl ether or methacrylic acid, themaleic anhydride constituting at least 20 mole percent of the copolymer.These materials are normally used at levels of from 0.5% to 10% byweight, more preferably form 0.75% to 8%, most preferably from 1% to 6%by weight of the composition.

[0246] Preferred optical brighteners are anionic in character, examplesof which are disodium4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′disulphonate, disodium 4,-4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium 4′,4″-bis-(2,4-dianilino-s-tri-azin-6ylamino)stilbene-2-sulphonate, disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,di-sodium 4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′disulphonate, di-so-dium4,4′-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylami-no)stilbene-2,2′disulphonate,sodium 2(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3, -triazole-2″-sulphonateand 4,4′-bis(2-sulphostyryl)biphenyl.

[0247] Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

[0248] Soil release agents useful in compositions of the presentinvention are conventionally copolymers or terpolymers of terephthalicacid with ethylene glycol and/or propylene glycol units in variousarrangements. Examples of such polymers are disclosed in U.S. Pat. No.4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymerin accordance with EP 0 272 033 has the formula:

(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25)[T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)

[0249] where PEG is —(OC₂H₄)O—, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

[0250] Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

[0251] The selected polyesters herein contain about 46% by weight ofdimethyl terephthalic acid, about 16% by weight of 1,2-propanediol,about 10% by weight ethylene glycol, about 13% by weight of dimethylsulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, andhave a molecular weight of about 3.000. The polyesters and their methodof preparation are described in detail in EP 311 342.

[0252] Softening Agents

[0253] Fabric softening agents can also be incorporated into laundrydetergent compositions in accordance with the present invention. Theseagents may be inorganic or organic in type. Inorganic softening agentsare exemplified by the smectite clays disclosed in GB-A-1400898 and inU.S. Pat. No. 5,019,292. Organic fabric softening agents include thewater insoluble tertiary amines as disclosed in GB-Al 514 276 and EP 0011 340 and their combination with mono C₁₂-C₁₄ quaternary ammoniumsalts are disclosed in EP-B-0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

[0254] Levels of smectite clay are normally in the range from 5% to 15%,more preferably from 8% to 12% by weight, with the material being addedas a dry mixed component to the remainder of the formulation. Organicfabric softening agents such as the water-insoluble tertiary amines ordilong chain amide materials are incorporated at levels of from 0.5% to5% by weight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

[0255] Polymeric Dye-Transfer Inhibiting Agents

[0256] The detergent compositions according to the present invention mayalso comprise from 0.001% to 10%, preferably from 0.01% to 2%, morepreferably form 0.05% to 1% by weight of polymeric dye-transferinhibiting agents. Said polymeric dye-transfer inhibiting agents arenormally incorporated into detergent compositions in order to inhibitthe transfer of dyes from colored fabrics onto fabrics washed therewith.These polymers have the ability of complexing or adsorbing the fugitivedyes washed out of dyed fabrics before the dyes have the opportunity tobecome attached to other articles in the wash.

[0257] Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

[0258] Addition of such polymers also enhances the performance of theenzymes according the invention.

[0259] The detergent composition according to the invention can be inliquid, paste, gels, bars or granular forms.

[0260] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. No. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1483591.

[0261] Granular compositions according to the present invention can alsobe in “compact form”, i.e. they may have a relatively higher densitythan conventional granular detergents, i.e. form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

[0262] The compositions of the invention may for example, be formulatedas hand and machine laundry detergent compositions including laundryadditive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

[0263] The following examples are meant to exemplify compositions forthe present invention, but are not necessarily meant to limit orotherwise define the scope of the invention.

[0264] In the detergent compositions, the abbreviated componentidentifications have the following meanings:

[0265] AExS: Sodium alkylether sulphate

[0266] LAS: Sodium linear C₁₂ alkyl benzene sulphonate

[0267] TAS: Sodium tallow alkyl sulphate

[0268] XYAS: Sodium C_(1X)-C_(1Y) alkyl sulfate

[0269] SS: Secondary soap surfactant of formula 2-butyl octanoic acid

[0270] 25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0271] 45EY: A C₁₄-C₁₅ predominantly linear primary alcohol condensedwith an average of Y moles of ethylene oxide

[0272] XYEZS: C_(1X)-C_(1Y) sodium alkyl sulfate condensed with anaverage of Z moles of ethylene oxide per mole

[0273] Nonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcoholwith an average degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbh

[0274] CFM: C₁₂-C₁₄ alkyl N-methyl glucamide

[0275] TFM: C₁₆-C₁₈ alkyl N-methyl glucamide

[0276] Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)

[0277] NaSKS-6: Crystalline layered silicate of formula d-Na₂Si₂O₅

[0278] Carbonate: Anhydrous sodium carbonate

[0279] Phosphate: Sodium tripolyphosphate

[0280] MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecularweight about 80,000

[0281] Polyacrylate: Polyacrylate homopolymer with an average molecularweight of 8,000 sold under the tradename PA30 by BASF GmbH

[0282] Zeolite A: Hydrated Sodium Aluminosilicate of formulaNa₁₂(AlO₂SiO₂)₁₂.27H₂O having a primary particle size in the range from1 to 10 micrometers

[0283] Citrate: Tri-sodium citrate dihydrate

[0284] Citric: Citric Acid

[0285] Perborate: Anhydrous sodium perborate monohydrate bleach,empirical formula NaBO₂.H₂O₂

[0286] PB4: Anhydrous sodium perborate tetrahydrate

[0287] Percarbonate: Anhydrous sodium percarbonate bleach of empiricalformula 2Na₂CO₃.3H₂O₂

[0288] TAED: Tetraacetyl ethylene diamine

[0289] CMC: Sodium carboxymethyl cellulose

[0290] DETPMP: Diethylene triamine penta (methylene phosphonic acid),marketed by Monsanto under the Tradename Dequest 2060

[0291] PVP: Polyvinylpyrrolidone polymer

[0292] EDDS: Ethylenediamine-N, N′-disuccinic acid, [S,S] isomer in theform of the sodium salt

[0293] Suds Suppressor: 25% paraffin wax Mpt 50° C., 17% hydrophobicsilica, 58% paraffin oil

[0294] Granular Suds suppressor: 12% Silicone/silica, 18% stearylalcohol, 70% starch in granular form

[0295] Sulphate: Anhydrous sodium sulphate

[0296] HMWPEO: High molecular weight polyethylene oxide

[0297] TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

[0298] A granular fabric cleaning composition in accordance with theinvention may be prepared as follows: Sodium linear C₁₂ alkyl 6.5benzene sulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodiumnitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boricacid 4.0 Perborate 18.0 Phenol sulphonate 0.1 Minors Up to 100

DETERGENT EXAMPLE II

[0299] A compact granular fabric cleaning composition (density 800 g/l)in accord with the invention may be prepared as follows: 45AS 8.0 25E3S2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citric acid3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0Percarbonate 22.0 EDDS 0.3 Granular suds suppressor 3.5 water/minors Upto 100%

DETERGENT EXAMPLE III

[0300] Granular fabric cleaning compositions in accordance with theinvention which are especially useful in the laundering of coloredfabrics were prepared as follows: LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS3.1 10.0 45E7 4.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0Carbonate — 10 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate2.5 — Sulphate 5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2Oxide/copolymer of vinyl- imidazole and vinyl- pyrrolidone Perborate 1.0— Phenol sulfonate 0.2 — Water/Minors Up to 100%

DETERGENT EXAMPLE IV

[0301] Granular fabric cleaning compositions in accordance with theinvention which provide “Softening through the wash” capability may beprepared as follows: 45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 —5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate5.0 3.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

DETERGENT EXAMPLE V

[0302] Heavy duty liquid fabric cleaning compositions in accordance withthe invention may be prepared as follows: I II LAS acid form — 25.0Citric acid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE78.0 — CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8 — Oleic acid — 1.0 Ethanol4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-alkyldimethyl — 3.0 hydroxy ethyl ammonium chloride Smectite clay — 5.0 PVP2.0 — Water/Minors Up to 100%

[0303] Uses in the Textile Industry

[0304] The pectate lyase enzyme of the present invention is useful inthe cellulosic fiber processing industry for the pretreatment or rettingof fibers from hemp, flax or linen.

[0305] The processing of cellulosic material for the textile industry,as for example cotton fiber, into a material ready for garmentmanufacture involves several steps: spinning of the fiber into a yarn;construction of woven or knit fabric from the yarn and subsequentpreparation, dyeing and finishing operations. Woven goods areconstructed by weaving a filling yarn between a series of warp yarns;the yarns could be two different types. Knitted goods are constructed byforming a network of interlocking loops from one continuous length ofyarn. The cellulosic fibers can also be used for non-woven fabric.

[0306] The preparation process prepares the textile for the properresponse in dyeing operations. The sub-steps involved in preparation are

[0307] a. Desizing (for woven goods) using polymeric size like e.g.starch, CMC or PVA is added before weaving in order to increase the warpspeed; This material must be removed before further processing.

[0308] b. Scouring, the aim of which is to remove non-cellulosicmaterial from the cotton fiber, especially the cuticle (mainlyconsisting of waxes) and primary cell wall (mainly consisting of pectin,protein and xyloglucan). A proper wax removal is necessary for obtaininga high wettability, being a measure for obtaining a good dyeing. Removalof the primary cell wall—especially the pectins—improves wax removal andensures a more even dyeing. Further this improves the whiteness in thebleaching process. The main chemical used in scouring is sodiumhydroxide in high concentrations, up to 70 g/kg cotton and at hightemperatures, 80-95° C.; and

[0309] c. Bleaching; normally the scouring is followed by a bleach usinghydrogen peroxide as the oxidizing agent in order to obtain either afully bleached (white) fabric or to ensure a clean shade of the dye.

[0310] A one step combined scour/bleach process is also used by theindustry. Although preparation processes are most commonly employed inthe fabric state; scouring, bleaching and dyeing operations can also bedone at the fiber or yarn stage.

[0311] The processing regime can be either batch or continuous with thefabric being contacted by the liquid processing stream in open width orrope form. Continuous operations generally use a saturator whereby anapproximate equal weight of chemical bath per weight of fabric isapplied to the fabric, followed by a heated dwell chamber where thechemical reaction takes place. A washing section then prepares thefabric for the next processing step. Batch processing generally takesplace in one processing bath whereby the fabric is contacted withapproximately 8-15 times its weight in chemical bath. After a reactionperiod, the chemicals are drained, fabric rinsed and the next chemicalis applied. Discontinuous pad-batch processing involves a saturatorwhereby an approximate equal weight of chemical bath per weight offabric is applied to the fabric, followed by a dwell period which in thecase of cold pad-batch might be one or more days.

[0312] Woven goods are the prevalent form of textile fabricconstruction. The weaving process demands a “sizing” of the warp yarn toprotect it from abrasion. Starch, polyvinyl alcohol (PVA), carboxymethylcellulose, waxes and acrylic binders are examples of typical sizingchemicals used because of availability and cost. The size must beremoved after the weaving process as the first step in preparing thewoven goods. The sized fabric in either rope or open width form isbrought in contact with the processing liquid containing the desizingagents. The desizing agent employed depends upon the type of size to beremoved. For PVA sizes, hot water or oxidative processes are often used.The most common sizing agent for cotton fabric is based upon starch.Therefore most often, woven cotton fabrics are desized by a combinationof hot water, the enzyme α-amylase to hydrolyze the starch and a wettingagent or surfactant. The cellulosic material is allowed to stand withthe desizing chemicals for a “holding period” sufficiently long toaccomplish the desizing. The holding period is dependent upon the typeof processing regime and the temperature and can vary from 15 minutes to2 hours, or in some cases, several days. Typically, the desizingchemicals are applied in a saturator bath which generally ranges fromabout 15° C. to about 55° C. The fabric is then held in equipment suchas a “J-box” which provides sufficient heat, usually between about 55°C. and about 100° C., to enhance the activity of the desizing agents.The chemicals, including the removed sizing agents, are washed away fromthe fabric after the termination of the holding period.

[0313] In order to ensure a high whiteness or a good wettability andresulting dyeability, the size chemicals and other applied chemicalsmust be thoroughly removed. It is generally believed that an efficientdesizing is of crucial importance to the following preparationprocesses: scouring and bleaching.

[0314] The scouring process removes much of the non-cellulosic compoundsnaturally found in cotton. In addition to the natural non-cellulosicimpurities, scouring can remove dirt, soils and residual manufacturingintroduced materials such as spinning, coning or slashing lubricants.The scouring process employs sodium hydroxide or related causticizingagents such as sodium carbonate, potassium hydroxide or mixturesthereof. Generally an alkali stable surfactant is added to the processto enhance solubilization of hydrophobic compounds and/or prevent theirredeposition back on the fabric. The treatment is generally at a hightemperature, 80° C.-100° C., employing strongly alkaline solutions, pH13-14, of the scouring agent. Due to the non-specific nature of chemicalprocesses not only are the impurities but the cellulose itself isattacked, leading to damages in strength or other desirable fabricproperties. The softness of the cellulosic fabric is a function ofresidual natural cotton waxes. The non-specific nature of the hightemperature strongly alkaline scouring process cannot discriminatebetween the desirable natural cotton lubricants and the manufacturingintroduced lubricants. Furthermore, the conventional scouring processcan cause environmental problems due to the highly alkaline effluentfrom these processes. The scouring stage prepares the fabric for theoptimal response in bleaching. An inadequately scoured fabric will needa higher level of bleach chemical in the subsequent bleaching stages.

[0315] The bleaching step decolorizes the natural cotton pigments andremoves any residual natural woody cotton trash components notcompletely removed during ginning, carding or scouring. The main processin use today is an alkaline hydrogen peroxide bleach. In many cases,especially when a very high whiteness is not needed, bleaching can becombined with scouring.

[0316] The following non-limiting examples illustrate the invention.

[0317] Materials and Methods

[0318] Strains and Donor Organism

[0319]Thermotoga maritima, DSM 3109.

[0320]B. subtilis DN1885 (Diderichsen, B., Wedsted, U., Hedegaard, L.,Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, which encodesalpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J.Bacteriol., 172, 4315-4321). Competent cells were prepared andtransformed as described by Yasbin, R. E., Wilson, G. A. and Young, F.E. (1975) Transformation and transfection in lysogenic strains ofBacillus subtilis: evidence for selective induction of prophage incompetent cells. J. Bacteriol, 121:296-304.

[0321]E. coli DH10B (Life Technologies Ltd, England).

[0322]B. subtilis MB1053-1. This strain is PL 2306 in which the pectatelyase gene Pel has been disrupted resulting in a pectate lyase negativestrain. The disruption was performed essentially as described in (Eds.A. L. Sonenshein, J. A. Hoch and Richard Losick (1993) Bacillus subtilisand other Gram-Positive Bacteria, American Society for microbiology, p.618).

[0323] Competent cells were prepared and transformed as described byYasbin, R. E., Wilson, G. A. and Young, F. E. (1975) Transformation andtransfection in lysogenic strains of Bacillus subtilis: evidence forselective induction of prophage in competent cells. J. Bacteriol,121:296-304.

[0324]B. subtilis PP289-5. This strain is described in U.S. Pat. No.5,843,720, example 1, step 2C, issued on Dec. 1, 1998.

[0325]B. licheniformis SJ3047. This strain is described in PCT Patentapplication WO 99/41358. Essentially this strain is a amylase negativerecombinant B. licheniformis strain.

[0326] Plasmids

[0327] pZErO-2 (Invitrogen, Calif., USA)

[0328] pMOL944:

[0329] This plasmid is a pUB110 derivative essentially containingelements making the plasmid propagatable in Bacillus subtilis, kanamycinresistance gene and having a strong promoter and signal peptide clonedfrom the amyL gene of B. licheniformis ATCC14580. The signal peptidecontains a Sacil site making it convenient to clone the DNA encoding themature part of a protein in-fusion with the signal peptide. This resultsin the expression of a Pre-protein directed towards the exterior of thecell.

[0330] The plasmid was constructed by means of conventional geneticengineering techniques briefly described in the following.

[0331] Construction of pMOL944:

[0332] The pUB 10 plasmid (McKenzie, T. et al., 1986, Plasmid 15:93-103)was digested with the unique restriction enzyme NciI. A PCR fragmentamplified from the amyL promoter encoded on the plasmid pDN1981 (P. L.Jørgensen et al., 1990, Gene, 96, p 37-41.) was digested with NciI andinserted in the NciI digested pUB110 to give the plasmid pSJ2624.

[0333] The two PCR primers used have the following sequences: #LWN54945′-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTG (SEQ ID NO:10) TATCTCAGC-3′#LWN5495 5′-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTT (SEQ ID NO:11)GTCGACCTGCAGAATGAGGCAGCAAGAAGAT-3′

[0334] The primer #LWN5494 inserts a NotI site in the plasmid.

[0335] The plasmid pSJ2624 was then digested with SacI and NotI and anew PCR fragment amplified on amyL promoter encoded on the pDN1981 wasdigested with SacI and NotI and this DNA fragment was inserted in theSacI-NotI digested pSJ2624 to give the plasmid pSJ2670.

[0336] This cloning replaces the first amyL promoter cloning with thesame promoter but in the opposite direction. The two primers used forPCR amplification have the following sequences: #LWN59385′-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTC (SEQ ID NO:12)GACCTGCAGAATGAGGCAGCAAGAAGAT-3′ #LWN59395′-GTCGGAGCTCTATCAATTGGTAACTGTATCTCA (SEQ ID NO:13) GC-3′

[0337] The plasmid pSJ2670 was digested with the restriction enzymesPstI and BclI and a PCR fragment amplified from a cloned DNA sequenceencoding the alkaline amylase SP722 (disclosed in the InternationalPatent Application published as WO 95/26397 which is hereby incorporatedby reference in its entirety) was digested with PstI and BclI andinserted to give the plasmid pMOL944. The two primers used for PCRamplification have the following sequence: #LWN78645′-AACAGCTGATCACGACTGATCTTTTAGCTTGGC (SEQ ID NO:14) AC-3′ #LWN79015′-AACTGCAGCCGCGGCACATCATAATGGGACAAA (SEQ ID NO:15) TGGG-3′

[0338] The primer #LWN7901 inserts a SacI site in the plasmid.

[0339] General Molecular Biology Methods

[0340] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989) Molecular cloning: A laboratory manual,Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”. John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990).

[0341] Enzymes for DNA manipulations were used according to thespecifications of the suppliers (e.g. restriction endonucleases, ligasesetc. are obtainable from New England Biolabs, Inc.).

[0342] Media

[0343] TY (as described in Ausubel, F. M. et al. (eds.) “Currentprotocols in Molecular Biology”. John Wiley and Sons, 1995).

[0344] LB agar (as described in Ausubel, F. M. et al. (eds.) “Currentprotocols in Molecular Biology”. John Wiley and Sons, 1995).

[0345] LBPG is LB agar supplemented with 0.5% Glucose and 0.05 Mpotassium phosphate, pH 7.0

[0346] BPX media is described in EP 0 506 780 (WO 91/09129).

[0347] The End Point Lyase Assay (at 235 nm), Pectate Units.

[0348] For determination of the β-elimination an assay measuring theincrease in absorbance at 235 nm was carried out using the substrate1.0% polygalacturonic acid sodium salt (Sigma P-1879) solubilised in 0.1M EPPS buffer pH 8. Incubation for 20 minutes at 70° C. The reaction isstopped by adding 5 volumes of 0.02 M H₃PO₄. For calculation of thecatalytic rate an increase of 5.2 Absorbency at 235 units per mincorresponds to formation of 1 μmol of unsaturated product (Nasuna andStarr (1966) J. Biol. Chem. Vol 241 page 5298-5306; and Bartling,Wegener and Olsen (1995) Microbiology Vol 141 page 873-881).

[0349] One Pectate Unit is the amount of enzyme resulting in formationof one micromole cleaved per minute at pH 8.0 and 70° C.

[0350] Genomic DNA Preparation

[0351] Strain Thermotoga maritima, DSM 3109, was propagated in Medium343 at 80° C., anaerobically as specified by DSMZ (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (German Collection ofMicroorganisms and Cell Cultures)). After propagation the cells wereharvested, and genomic DNA isolated by the method described by Pitcheret al. (Pitcher, D. G., Saunders, N. A., Owen, R. J. (1989). Rapidextraction of bacterial genomic DNA with guanidium thiocyanate. Lett.Appl. Microbiol., 8,151-156).

[0352] The following examples illustrate the invention.

EXAMPLE 1

[0353] Cloning, Expression, Purification and Characterization of PectateLyase from Thermotoga maritima, DSM 3109

[0354] The pectate lyase encoded on the genome of Thermotoga maritima,(DSM 3109) (vide supra, represented by amino acid sequence SEQ ID NO:1)encoding DNA sequence of the invention was cloned as described below.

[0355] Genomic DNA of Thermotoga maritima, (DSM 3109) was used astemplate for PCR amplification using the primers #171130 and #171131yielding a fragment of 1.0 kbp. The fragment was isolated on a 0.7%agarose gel and digested by the restriction enzymes SacI and NotI.

[0356] Primers: #171130: AAA CCG CGG CAT CTC TCA ATG ACA AAC (SEQ IDNO:16) CTG TGG G (SacII) #171131: AAA GCG GCC GCT GAG CCG TAT TTA GTT(SEQ ID NO:17) CTT CAA ACA CC (NotI)

[0357] The isolated DNA fragment was ligated to the Sacil and NotIdigested plasmid pMOL944 (4.8 kbp), and the ligation mixture was usedfor transform Bacillus subtilis DN1885 (Diderichsen, B., Wedsted, U.,Hedegaard, L., Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, whichencodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillusbrevis. J. Bacteriol., 172, 4315-4321). Transformed cells from wereplated on LB-agar containing 10 mM Potassium phosphate buffer pH 7.0,0.4% glucose, 10 μg/ml kanamycin. The plated cells were incubated 16hours at 37° C.

[0358] Several clones were re-streaked on fresh agar plates and alsogrown in liquid TY cultures with 10 μg/ml kanamycin and incubatedovernight at 37° C. Next day 1 ml of cells were used to isolate plasmidfrom the cells using the Qiaprep Spin Plasmid Miniprep Kit #27106according to the manufacturers recommendations for B. subtilis plasmidpreparations. One correct construct was saved as the clone JA855. Thisplasmid DNA (pJA855) was used as template for DNA sequencing. The DNAsequence thus revealed is represented by DNA sequence SEQ ID NO:2. Thissequence is a fusion between the DNA encoding the signal peptide clonedfrom the amyL gene of B. licheniformis ATCC14580 and the DNA encodingthe mature part of the pectate lyase of Thermotoga maritima, whereinpositions 1 to 87 are the AmyL-signal; positions 88 to 1107 encode theThermotoga maritima pectate lyase mature part, and positions 1108 to1149 are vector pMOL944 DNA.

[0359] The derived protein sequence of SEQ ID NO:2 is represented in SEQID NO:3 where positions 1 to 29 are the AmyL signal peptide, positions30 to 369 are the mature pectate lyase, and positions 370 to 383 theexpressed vector.

[0360] The cloned DNA sequence was expressed in B. subtilis byfermenting the JA855 cells in BP-X media containing 10 μg/ml ofkanamycin at 37° C. for 5 days at 300 rpm.

EXAMPLE 2

[0361] Purification and Characterisation of Pectate Lyase Cloned fromThermotoga maritima (JA855)

[0362] The clone JA855 obtained as described in example 1 was incubatedin 2200 ml of BPX containing mg/ml kanamycin from shake flasks with afinal pH of 7.2.

[0363] The fermentation medium was diluted with one volume of water andflocculated using cationic flocculation agent C521 (10% solution) and0.1% solution of anionic agent A130: To 4000 ml of broth was added 170ml of C521 (10%) simultaneous with 380 ml of A130 under stirring at roomtemperature. The flocculated material was separated by centrifugationusing a Sorval RC 3B centrifuge at 4,500 rpm for 30 minutes. Thesupernatant was clarified using Whatman glass filter number F. In totalwas obtained 4200 ml of clear solution containing 100,000 Pectate Units.

[0364] The liquid was concentrated into 400 ml using filtronultrafiltration with a MW cut-off of 10 kDa.

[0365] For obtaining a pure enzyme 2 ml of this partial pure enzyme wasapplied to a size chromatography (Superdex 200) column equilibrated with0.1 M sodium acetate, pH 6.0. The pectate lyase eluted as a single peakwith a MW of 38 kDa in SDS-PAGE and with a specific activity of 50Pectate Units per mg protein.

[0366] The cloned pectate lyase of the invention was used for raisingrabbit antiserum.

[0367] After electroblotting of this band the N-terminal was determinedas:

[0368] ASLNDKPVGFASVP

[0369] This is in agreement with the amino acid sequence shown in SEQ IDNO:3 deduced from the DNA sequence shown in SEQ ID NO:2 with a 28 aminoacid prosequence. The calculated MW from the deduced sequence was 38 kDaand the calculated pI was 5. The molar extinction coefficient at 280 nmwas 37,460.

[0370] The β-transelimination activity (using the lyase assay at 235 nm)at different pH values was determined as steady state kinetic at 70° C.,substrate 1.0% polygalacturonic acid sodium salt (Sigma P-1879). Therelative rate is calculated as percentage of the optimum activity, thefollowing result was obtained: PH % Activity 4.5 1 5.0 1 5.4 4 6.9 7 7.411 7.8 9 8.3 19 8.5 11 9.3 34 9.9 81 10.2 100

[0371] Correspondingly, the relative activity at different temperatures(at pH 9; 0.39 millimoles CaCl₂; substrate 1.0% polygalacturonic acidsodium salt (Sigma P-1879)) was found: Temp. ° C. % Activity 70 38 80 5490 76 100 100

[0372] Correspondingly, the relative activity at different temperatures(at pH 8; 0.39 mmol CaCl₂; substrate 1.0% polygalacturonic acid sodiumsalt (Sigma P-1879)) was found: Temp. ° C. % Activity 70 22 80 48 90 81100 100

[0373] CaCl₂ dependency at pH 8.0 and 70° C.: Optimum activity wasobtained using between 0.1 and 0.6 millimoles CaCl₂. Excess EDTAinhibited the catalytic activity.

[0374] DSC in sodium acetate buffer at pH 6.0 showed a meltingtemperature around 98° C.

EXAMPLE 3

[0375] Cloning and Verification of Thermotoga maritima Pectate Lyase inE. coli

[0376] Cloning of the Thermotoga maritima Pectate Lyase

[0377] The pectate lyase Thermotoga maritima, DSM 3109, was cloned byPCR cloning applying the following primers:GGGAATTCTTACTGAGCCGTATTTAGTTC (SEQ ID NO:18) andCCGGATCCAGTAGGGAGGGATGCTCATG (SEQ ID NO:19) on purified chromosomal DNA.The purified PCR fragment was digested with EcoRI and BamHI, cloned intopZErO-2 (Invitrogen, Calif., USA) restricted with EcoRI and BamHI andtransformed into E. coli DH10B (Life Technologies Ltd, England). Severalclones were sequenced and one clone with the right sequence encoding theThermotoga maritima pectate lyase was given the name PEC1038. This DNAsequence is represented as the SEQ ID NO:4 and the derived proteinsequence is represented as the SEQ ID NO:5. Molecular biology wasconducted using methods known to persons skilled in the art.

[0378] Functional Analysis:

[0379] The pectate lyase of Thermotoga maritima was functionallycharacterized as follows: The PEC1038 clone was inoculated into TYmedium with 25 μg/ml kanamycin, grown to OD₄₉₀˜0.6 when isopropylβ-D-thiogalactopyranoside (Sigma) was added to the final concentrationof 1 mM. The growth was continued at 37° C. overnight before sampleswere tested for pectinase activity.

[0380] Fluorescence polarisation (PCT/DK99/00112) was applied in orderto monitor the pectinase activity, as this technology is compatible withthe elevated temperature. Assay mixes consisting of 100 μl overnightcultures (PEC1038 was used as positive sample and DH10B as negativecontrol) and 400 μl assay buffer (50 mM Hepes pH 8, 25 μg/ml fluoresceinlabelled pectin (DE 3%) (PCT/DK99/00112) and 1 mM CaCl₂) was incubatedin Eppendorf tubes placed in heating blocks set at various temperatures(See Table 1). Mineral oil was applied on top of the assay mixes inorder to avoid evaporation. The Bacillus agaradhaerens pectate lyase(cf. WO 99/27084, SEQ ID NO:2) was included as positive control and wasapplied at the final concentration of 5.5 μg/ml.

[0381] Values in Table 1 are given as relative changes in fluorescencepolarisation value corrected for the background (DH10B sample). As canbe seen, end-point reaction resulted in a decrease in the polarisationvalue of approximately 23%. The PEC1038 samples did only reach end-pointreaction at elevated temperatures (80° C. and 90° C.) as supposed to 9%decrease at 60° C. after 3 hours incubation which demonstrates that theThermotoga pectate lyase is relatively more active at 80° C. and 90° C.compared to 60° C. The experiment also demonstrates that the pectatelyase from Thermotoga maritima is more active at 90° C. than at 80° C.(See data for 1 and 2 hours incubation). 60° C. 70° C. 80° C. 90° C. 1hour PEC1038 4% 1% 8% 16% B. agradhaerens 23% pectate lyase 2 hoursPEC1038 8% 6% 9% 20% B. agaradhaerens 23% pectate lyase 3 hours PEC10389% 19% 24% 21% B. agaradhaerens 24% pectate lyase

[0382] Table 1: Activity of PEC1038 and B. agaradhaerens pectate lyase,shown as relative decreases given in percentage of the polarisationvalue of the negative control DH10B). Samples were incubated at 60° C.,70° C., 80° C. and 90° C. for 1, 2 and 3 hours.

EXAMPLE 4

[0383] Cloning of Native Pectate Lyase from Thermotoga maritima, DSM3109

[0384] The pectate lyase encoded on the genome of Thermotoga maritima,DSM 3109 (vide supra, represented by amino acid sequence SEQ ID NO:1)encoding DNA sequence of the invention was cloned as described below.

[0385] Genomic DNA of Thermotoga maritima, DSM 3109, was used astemplate for PCR amplification. The oligonucleotides #185245 and #186757were used in a PCR reaction in HiFidelity™ PCR buffer (BoehringerMannheim, Germany) supplemented with 200 μM of each dNTP, 2.6 units ofHiFidelity™ Expand enzyme mix and 200 pmol of each primer.

[0386] Primers: #185245: 5′-CAT TCT GCA GCC GCG GCA TCT CTC (SEQ IDNO:20) AAT GAC AAA CCT GTG GG-3′ (SacII) #186757: 5′-CAT CAT GGA TCC GCGGCC GCT TAT (SEQ ID NO:21) CAC TGA GCC GTA TTT AGT TCT TCA AAC-3′ (NotI)

[0387] The PCR reaction was performed using a DNA thermal cycler(Landgraf, Germany). One incubation at 94° C. for 1 min followed by tencycles of PCR performed using a cycle profile of denaturation at 94° C.for 15 sec, annealing at 60° C. for 60 sec, and extension at 72° C. for120 sec, followed by twenty cycles of denaturation at 94° C. for 15 sec,60° C. for 60 sec and 72° C. for 120 sec (at this elongation step 20 secare added every cycle). Five μl aliquots of the amplification productwas analysed by electrophoresis in 0.7% agarose gels (NuSieve, FMC). Theappearance of a DNA fragment size 1.0 kb indicated proper amplificationof the gene segment.

[0388] Subcloning of PCR Fragment:

[0389] Forty five μl aliquots of the PCR products generated as describedabove were purified using QIAquick PCR purification kit (Qiagen, USA)according to the manufacturer's instructions. The purified DNA waseluted in 50 μl of 10 mM Tris-HCl, pH 8.5.

[0390] 5 μg of pMOL944 and twenty five μl of the purified PCR fragmentwas digested with SacI and NotI, electrophoresed in 0.7% agarose gels(NuSieve, FMC), the relevant fragments were excised from the gels, andpurified using QIAquick Gel extraction Kit (Qiagen, USA) according tothe manufacturer's instructions. The isolated PCR DNA fragment was thenligated to the SacI-NotI digested and purified pMOL944. The ligation wasperformed overnight at 16° C. using 0.5 μg of each DNA fragment, 1 U ofT4 DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

[0391] The ligation mixture was used for transform Bacillus subtilisMB1053-1. Transformed cells from were plated on LB-agar containing 10 mMpotassium phosphate buffer pH 7.0, 0.4% glucose, 10 μg/ml kanamycin. Theplated cells were incubated for 16 hours at 37° C.

[0392] Several clones were re-streaked on fresh agar plates and alsogrown in liquid TY cultures with 10 μg/ml kanamycin and incubatedovernight at 37° C. Next day 1 ml of cells were used to isolate plasmidfrom the cells using the Qiaprep Spin Plasmid Miniprep Kit #27106according to the manufacturers recommendations for B. subtilis plasmidpreparations. One correct construct was saved as the clone MB1083. Thisplasmid DNA, pMB1083 was used as template for DNA sequencing. The DNAsequence thus revealed is represented by DNA sequence SEQ ID NO:6. Thissequence is a fusion between the DNA encoding the signal peptide clonedfrom the amyL gene of B. licheniformis ATCC14580 and the DNA encodingthe mature part of the pectatelyase of Thermotoga maritima, whereinpositions 1 to 84 represent the AmyL-signal and positions 85 to 1107 arethe Thermotoga maritima pectate lyase mature part.

[0393] The derived protein sequence of SEQ ID NO:6 is represented in SEQID NO:7 where positions 1 to 29 are the AmyL signal peptide andpositions 30 to 369 are the mature pectate lyase.

[0394] The cloned DNA sequence was expressed in B. subtilis byfermenting the MB1083 cells in BP-X media containing 10 μg/ml ofkanamycin, at 37° C. for 5 days at 300 rpm.

EXAMPLE 5

[0395] Expression of Thermotoga maritima pectate lyase in B.licheniformis

[0396] Plasmid pMB1083 was used to transform competent cells of B.subtilis PP289-5 (dal-, pLS20, pBC16; U.S. Pat. No. 5,843,720, example1, step 2C) selecting kanamycin (10 μg/ml) and tetracycline (5 pg/ml)resistance at 30° C. on LBPG agar plates supplemented with D-alanine(100 μg/ml). One transformant was kept, MB1101.

[0397] The donor strain MB1101 was used to transfer its plasmid into B.licheniformis by is conjugation, essentially as described in U.S. Pat.No. 5,843,720, example 1, step 2D. Transconjugants were selected on LBPG10 μg/ml Kanamycin plates. One transconjugant was kept, MB1105.

[0398] MB1105 was grown overnight in LB media, plasmid DNA was isolatedand characterized, the characterization revealed that the originalplasmid, pMB1083, had been established in B. licheniformis. Therecombinant Thermotoga maritima pectate lyase expressed from MB1105 wasexpressed, purified and characterized as described below.

EXAMPLE 6

[0399] Purification and Characterisation of Pectate Lyase Cloned fromThermotoga maritima and Expressed in Bacillus licheniformis (cloneMB1105)

[0400] The clone MB1105 obtained as described in example 6 was incubatedin shake flasks using the substrate PS1 with 10 μg/l kanamycin and total3100 ml fermentation broth was obtained from the shake flasks.

[0401] The fermentation medium was adjusted to pH 7.5 and 31 ml of 50%CaCl₂ was added. Then 31 ml of 11% freshly made solution of sodiumaluminates was added using a pH titration and 20% formic acid forkeeping the pH at 7.5. Finally the cells were flocculated using cationicflocculation agent C521 (10% solution) and 0.1% solution of anionicagent A130: 78 ml of C521 (10%) was added simultaneously with 233 ml ofA130 under stirring at room temperature. The flocculated material wasseparated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpmfor 20 minutes. The supernatant was clarified using Whatman glass filternumber F. In total was obtained 2500 ml of clear solution containing280,000 Pectate Units.

[0402] The liquid was concentrated into 360 ml, using filtron ultrafiltration with a MW cut off of 10 kDa. The solution was diluted to 1450ml using ionized water and applied to a Q-Sepharose column equilibratedwith 25 mM Tris pH 7.5. The pectate lyase was eluted from the columnusing a sodium chloride gradient. For obtaining a pure enzyme 2 ml ofthis partial pure enzyme was applied to a size chromatography (Superdex200) column equilibrated with 0.1 M sodium acetate pH 6.0. The pectatelyase eluted as a single peak with a MW of 38 kDa in SDS-PAGE.

EXAMPLE 7

[0403] Cloning, Expression of a Variant of Pectate Lyase from Thermotogamaritima, DSM 3109

[0404] The DNA sequence of the Thermotoga maritima pectate lyase wasaltered in such a way that three cysteine codons were changed into threeother amino acids, the resulting DNA sequence and derived proteinsequence are found in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0405] Plasmid DNA pJA855 was used as template for PCR amplification.The oligonucleotides #185245 and #186339 were used in a PCR reaction inHiFidelity™ PCR buffer (Boehringer Mannheim, Germany) supplemented with200 μM of each dNTP, 2.6 units of HiFidelity™ Expand enzyme mix and 200pmol of each primer. In another PCR reaction the oligonucleotides#186757 and #186340 were used in a PCR reaction in HiFidelity™ PCRbuffer (Boehringer Mannheim, Germany) supplemented with 200 pM of eachdNTP, 2.6 units of HiFidelity™ Expand enzyme mix and 200 pmol of eachprimer. The two resulting PCR fragments were purified using QIAquick PCRpurification kit (Qiagen, USA) according to the manufacturer'sinstructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH8.5.

[0406] These two PCR fragments were assembled in a third PCR reactioncalled a SOE PCR, equimolar amounts of the two PCR fragments were set upin a PCR reaction in HiFidelity™ PCR buffer (Boehringer Mannheim,Germany) supplemented with 200 μM of each dNTP, 2.6 units of HiFidelity™Expand enzyme mix and 200 pmol of each of the primers #185245 and#186757.

[0407] Primers: #185245: 5′-CAT TCT GCA GCC GCG GCA TCT CTC (SEQ IDNO:22) AAT GAC AAA CCT GTG GG-3′ (SacII) #186339: 5′-CCA ACG AGG GAG ACCTTG TCG TGA (SEQ ID NO:23) TCC ACA AAT TTG YTC CAG GAA ACA GTG ATG TAGTTT GAG TAT TTT TTA ATA TCC ACT GCA CCA TCG TTG CCG TTG ACG AAG GTA WYGTGG TCG-3′ #186340: 5′-GGA TCA CGA CAA GGT CTC CCT CGT (SEQ ID NO:24)TGG TTC CTC CGA CAA AGA AGA TCC GGA ACA GGC AGG GCA GGC TTA CAA GGT CACGTA CCA CCA TAA CTA CTT CAA GAA CCT GAT TCA GAG-3′ #186757: 5′-CAT CATGGA TCC GCG GCC GCT TAT (SEQ ID NO:25) CAC TGA GCC GTA TTT AGT TCT TCAAAC- 3′ (NotI)

[0408] The PCR reactions were performed using a DNA thermal cycler(Landgraf, Germany). One incubation at 94° C. for 1 min followed by tencycles of PCR performed using a cycle profile of denaturation at 94° C.for 15 sec, annealing at 60° C. for 60 sec, and extension at 72° C. for120 sec, followed by twenty cycles of denaturation at 94° C. for 15 sec,60° C. for 60 sec and 72° C. for 120 sec (at this elongation step 20 secare added every cycle). Five μl aliquots of the amplification productwas analysed by electrophoresis in 0.7% agarose gels (NuSieve, FMC).

[0409] Subcloning of PCR Fragment:

[0410] Forty five μl aliquots of the third PCR product (from the SOEPCR) generated as described above were purified using QIAquick PCRpurification kit (Qiagen, USA) according to the manufacturer'sinstructions. The purified DNA was eluted in 50 μl of 10 mM Tris-HCl, pH8.5.

[0411] 5 μg of pMOL944 and twenty five μl of the purified PCR fragmentwas digested with SacI and NotI, electrophoresed in 0.7% agarose gels(NuSieve, FMC), the relevant fragments were excised from the gels, andpurified using QIAquick Gel extraction Kit (Qiagen, USA) according tothe manufacturer's instructions. The isolated PCR DNA fragment was thenligated to the SacI-NotI digested and purified pMOL944. The ligation wasperformed overnight at 16° C. using 0.5 μg of each DNA fragment, 1 U ofT4 DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

[0412] The ligation mixture was used for transforming Bacillus subtilisMB1053-1. Transformed cells were plated on LB-agar containing 10 mMpotassium phosphate buffer pH 7.0, 0.4% glucose, 10 μg/ml kanamycin. Theplated cells were incubated for 16 hours at 37° C.

[0413] Several clones were re-streaked on fresh agar plates and alsogrown in liquid TY cultures with 10 μg/ml kanamycin and incubatedovernight at 37° C. Next day 1 ml of cells were used to isolate plasmidfrom the cells using the Qiaprep Spin Plasmid Miniprep Kit #27106according to the manufacturers recommendations for B. subtilis plasmidpreparations. One construct was saved as the clone MB1081. This plasmidDNA, pMB1081, was used as template for DNA sequencing. The DNA sequencethus revealed is represented by DNA sequence SEQ ID NO:8. This sequenceis a fusion between the DNA encoding the signal peptide cloned from theamyL gene of B. licheniformis ATCC14580 and the DNA encoding the maturepart of the pectate lyase of Thermotoga maritima, wherein three of thecysteine codons are altered to three other amino acid codons. In thesequence, positions 1 to 84 are the AmyL-signal, and positions 85 to1107 are the Thermotoga maritima pectate lyase mature part with threecysteine codons altered.

[0414] The derived protein sequence of SEQ ID NO:8 is represented in SEQID NO:9 wherein positions 1-29 are AmyL signal peptide; postions 30-369are the mature pectate lyase with three cysteins altered.

[0415] The cloned DNA sequence was expressed in B. subtilis byfermenting the MB1081 cells in BP-X media containing 10 μg/ml ofkanamycin, at 37° C. for 5 days at 300 rpm.

EXAMPLE 8

[0416] Expression of Thermotoga maritima Pectate Lyase in B.licheniformis

[0417] Plasmid pMB1081 was used to transform competent cells of B.subtilis PP289-5 (dal-, pLS20, pBC16; U.S. Pat. No. 5,843,720, example1, step 2C) selecting kanamycin (10 μg/ml) and tetracycline (5 μg/ml)resistance at 30° C. on LBPG agar plates supplemented with D-alanine(100 μg/ml). One transformant was kept, MB1000.

[0418] The donor strain MB1000 was used to transfer its plasmid into B.licheniformis by conjugation, essentially as described in U.S. Pat. No.5,843,720, example 1, step 2D. Transconjugants were selected on LBPG 10μg/ml Kanamycin plates. One transconjugant was kept, MB1104.

[0419] MB1104 was grown overnight in LB media, plasmid DNA was isolatedand characterized, the characterization revealed that the originalplasmid, pMB1081, had been established in B. licheniformis. Therecombinant Thermotoga maritima pectate lyase expressed from MB1104 wasexpressed, purified and characterised as described below.

EXAMPLE 9

[0420] Purification and Characterisation of Pectate Lyase Cloned fromThermotoga maritima, Site Directed Variant with 3 Cysteines Substituted,and Expressed in Bacillus licheniformis (Clone MB1104)

[0421] The clone MB1104 obtained as described in example 8 was incubatedin 500 ml shake flasks containing 100 ml BP-X media with 10 μg/lkanamycin a total of 3300 ml fermentation broth was obtained from shakeflasks.

[0422] The fermentation medium was adjusted to pH 7.5 and 33 ml 50%CaCl₂ was added. Then 33 ml of 11% freshly made solution of sodiumaluminates was added using a pH titration and 20% formic acid forkeeping the pH at 7.5. Finally the cells were flocculated using cationicflocculation agent C521 (10% solution) and 0.1% solution of anionicagent A130: 83 ml of C521 (10%) was added simultaneously with 248 ml ofA130 under stirring at room temperature. The flocculated material wasseparated by centrifugation using a Sorval RC 3B centrifuge at 4,500 rpmfor 20 minutes. The supernatant was filtrated using Whatman glassfilters GF/D and F. The clear sterile solution was concentrated on aFiltron with a MW cut-off at 10 kDa, and the concentrate was dilutedwith ion-free water for finally to obtain a solution with a conductivityof 3 mSi in a volume of 1000 ml and the pH adjusted to 7.5. This wasthen applied to Q-Sepharose column equilibrated with 25 mM Tris pH 7.5.The pectate lyase bound to the ion-exchange column and was eluted usinga NaCl gradient. The pure enzyme was 90% pure in SDS-PAGE with a mainband at 38 kDa. The enzyme was further purified using Sephadex.

[0423] The purified pectate lyase from MB1104 was analyzed for itsactivity in different buffers representing different pH. The buffersused were: Na-MES 0.1 M pH 6.0; Na-MOPS 0.1 M pH 6.5, Na-MOPS 0.1 M pH7.0; Phosphate 0.1 M pH 7.5; EPPS 0.1 M pH 8.0; EPPS 0.1 M pH 8.5;Na.glycine 0.1 M pH 9.0; Na.glycine 0.1 M pH 9.5; Na.glycine 0.1 M pH10.0 and Na.glycine 0.1 M pH 10.5. Pectate lyase activity was determinedas described above in the section “The End Point Lyase assay (at 235nm), Pectate Units.” The incubation temperature in this investigationwas 70° C.

[0424] The β-transelimination activity (using the lyase assay at 235 nm)at different pH values was determined as steady state kinetic at 70° C.,(at pH 8; 0.68 millimoles CaCl₂; substrate 1.0% polygalacturonic acidsodium salt (Sigma P-1879)). The relative rate is calculated aspercentage of the optimum activity, the following result was obtained:pH % Activity 6.0 9.0 6.5 13.4 7.0 18.6 7.5 63.2 8.0 33.9 8.5 47.0 9.029.2 9.5 43.3 10 86.6 10.5 100

[0425] The purified pectate lyase from MB1104 was analyzed for itsactivity under different temperatures the bufer system was 0.1 M EPPS pH8.0 and temperatures investigated was: 70° C., 80° C., 90° C. and 95° C.Pectate lyase activity was determined as described above in the section“The End Point Lyase assay (at 235 nm), Pectate Units.” In the tablebelow the activity measured at 95° C. was set to a 100%.

[0426] Similar to the determination of the pH profile, the relativeactivity at different temperatures (at pH 8; 0.68 millimoles CaCl₂;substrate 1.0% polygalacturonic acid sodium salt (Sigma P-1879)) wasfound: Temp. ° C. % Activity 70 39 80 53 90 69 95 100

[0427]

1 25 1 340 PRT Thermotoga maritima 1 Ser Leu Asn Asp Lys Pro Val Gly PheAla Ser Val Pro Thr Ala Asp 1 5 10 15 Leu Pro Glu Gly Thr Val Gly GlyLeu Gly Gly Glu Ile Val Phe Val 20 25 30 Arg Thr Ala Glu Glu Leu Glu LysTyr Thr Thr Ala Glu Gly Lys Tyr 35 40 45 Val Ile Val Val Asp Gly Thr IleVal Phe Glu Pro Lys Arg Glu Ile 50 55 60 Lys Val Leu Ser Asp Lys Thr IleVal Gly Ile Asn Asp Ala Lys Ile 65 70 75 80 Val Gly Gly Gly Leu Val IleLys Asp Ala Gln Asn Val Ile Ile Arg 85 90 95 Asn Ile His Phe Glu Gly PheTyr Met Glu Asp Asp Pro Arg Gly Lys 100 105 110 Lys Tyr Asp Phe Asp TyrIle Asn Val Glu Asn Ser His His Ile Trp 115 120 125 Ile Asp His Cys ThrPhe Val Asn Gly Asn Asp Gly Ala Val Asp Ile 130 135 140 Lys Lys Tyr SerAsn Tyr Ile Thr Val Ser Trp Cys Lys Phe Val Asp 145 150 155 160 His AspLys Val Ser Leu Val Gly Ser Ser Asp Lys Glu Asp Pro Glu 165 170 175 GlnAla Gly Gln Ala Tyr Lys Val Thr Tyr His His Asn Tyr Phe Lys 180 185 190Asn Cys Ile Gln Arg Met Pro Arg Ile Arg Phe Gly Met Ala His Val 195 200205 Phe Asn Asn Phe Tyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn 210215 220 Val Phe Pro Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val His225 230 235 240 Val Glu Gly Asn Tyr Phe Met Gly Tyr Gly Ala Val Met AlaGlu Ala 245 250 255 Gly Ile Ala Phe Leu Pro Thr Arg Ile Met Gly Pro ValGlu Gly Tyr 260 265 270 Leu Thr Leu Gly Glu Gly Asp Ala Lys Asn Glu PheTyr Tyr Cys Lys 275 280 285 Glu Pro Glu Val Arg Pro Val Glu Glu Gly LysPro Ala Leu Asp Pro 290 295 300 Arg Glu Tyr Tyr Asp Tyr Thr Leu Asp ProVal Gln Asp Val Pro Lys 305 310 315 320 Ile Val Val Asp Gly Ala Gly AlaGly Lys Leu Val Phe Glu Glu Leu 325 330 335 Asn Thr Ala Gln 340 2 1149DNA Artificial Sequence Synthetic 2 atg aaa caa caa aaa cgg ctt tac gcccga ttg ctg acg ctg tta ttt 48 Met Lys Gln Gln Lys Arg Leu Tyr Ala ArgLeu Leu Thr Leu Leu Phe -25 -20 -15 gcg ctc atc ttc ttg ctg cct cat tctgca gcc gcg gca tct ctc aat 96 Ala Leu Ile Phe Leu Leu Pro His Ser AlaAla Ala Ala Ser Leu Asn -10 -5 -1 1 gac aaa cct gtg gga ttt gca tcc gtaccg acg gcg gat tta ccg gag 144 Asp Lys Pro Val Gly Phe Ala Ser Val ProThr Ala Asp Leu Pro Glu 5 10 15 ggc aca gtt ggt gga ttg ggt ggt gag atcgtt ttc gtc aga aca gcg 192 Gly Thr Val Gly Gly Leu Gly Gly Glu Ile ValPhe Val Arg Thr Ala 20 25 30 35 gaa gaa ctg gag aaa tac aca acg gca gaagga aag tac gta ata gtc 240 Glu Glu Leu Glu Lys Tyr Thr Thr Ala Glu GlyLys Tyr Val Ile Val 40 45 50 gtt gat gga acg atc gtt ttt gaa cca aag agagaa att aaa gtt ctt 288 Val Asp Gly Thr Ile Val Phe Glu Pro Lys Arg GluIle Lys Val Leu 55 60 65 tca gac aaa acg atc gtg gga ata aac gat gca aagata gtc ggt gga 336 Ser Asp Lys Thr Ile Val Gly Ile Asn Asp Ala Lys IleVal Gly Gly 70 75 80 ggt ctt gtg ata aag gat gcc cag aat gtg atc ata agaaat att cat 384 Gly Leu Val Ile Lys Asp Ala Gln Asn Val Ile Ile Arg AsnIle His 85 90 95 ttt gag ggc ttt tac atg gag gac gat cct cgg ggt aag aagtat gat 432 Phe Glu Gly Phe Tyr Met Glu Asp Asp Pro Arg Gly Lys Lys TyrAsp 100 105 110 115 ttc gac tat atc aac gtg gaa aat tct cat cat atc tggatc gac cac 480 Phe Asp Tyr Ile Asn Val Glu Asn Ser His His Ile Trp IleAsp His 120 125 130 tgt acc ttc gtc aac ggc aac gat ggt gca gtg gat attaaa aaa tac 528 Cys Thr Phe Val Asn Gly Asn Asp Gly Ala Val Asp Ile LysLys Tyr 135 140 145 tca aac tac atc act gtt tcc tgg tgt aaa ttt gtg gatcac gac aag 576 Ser Asn Tyr Ile Thr Val Ser Trp Cys Lys Phe Val Asp HisAsp Lys 150 155 160 gtc tcc ctc gtt ggt tcc tcc gac aaa gaa gat ccg gaacag gca ggg 624 Val Ser Leu Val Gly Ser Ser Asp Lys Glu Asp Pro Glu GlnAla Gly 165 170 175 cag gct tac aag gtc acg tac cac cat aac tac ttc aagaac tgt att 672 Gln Ala Tyr Lys Val Thr Tyr His His Asn Tyr Phe Lys AsnCys Ile 180 185 190 195 cag aga atg ccc aga att aga ttt gga atg gca cacgtg ttc aat aac 720 Gln Arg Met Pro Arg Ile Arg Phe Gly Met Ala His ValPhe Asn Asn 200 205 210 ttc tac agc atg ggc ctg aga aca ggt gtc tct ggaaac gtc ttc ccc 768 Phe Tyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly AsnVal Phe Pro 215 220 225 att tac ggt gtt gct tca gcg atg gga gcg aaa gtccac gtt gaa gga 816 Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val HisVal Glu Gly 230 235 240 aac tac ttc atg gga tac ggt gct gtg atg gca gaggcg gga att gcg 864 Asn Tyr Phe Met Gly Tyr Gly Ala Val Met Ala Glu AlaGly Ile Ala 245 250 255 ttc ctt ccc acc aga atc atg ggt ccc gtg gaa ggttat ctg acg ctc 912 Phe Leu Pro Thr Arg Ile Met Gly Pro Val Glu Gly TyrLeu Thr Leu 260 265 270 275 ggt gaa gga gat gca aag aat gaa ttt tac tactgt aaa gaa cct gaa 960 Gly Glu Gly Asp Ala Lys Asn Glu Phe Tyr Tyr CysLys Glu Pro Glu 280 285 290 gtg cgt cct gtt gag gaa gga aaa ccc gct ctcgat cca cgc gag tac 1008 Val Arg Pro Val Glu Glu Gly Lys Pro Ala Leu AspPro Arg Glu Tyr 295 300 305 tac gat tac acg ctt gat cca gtt caa gat gttcca aaa atc gtt gta 1056 Tyr Asp Tyr Thr Leu Asp Pro Val Gln Asp Val ProLys Ile Val Val 310 315 320 gat gga gca gga gca ggg aaa ctg gtg ttt gaagaa cta aat acg gct 1104 Asp Gly Ala Gly Ala Gly Lys Leu Val Phe Glu GluLeu Asn Thr Ala 325 330 335 cag cgg ccg ccc cgg cat tgc cag tcg ggg atatta aaa aga gta 1149 Gln Arg Pro Pro Arg His Cys Gln Ser Gly Ile Leu LysArg Val 340 345 350 3 383 PRT Artificial Sequence Synthetic 3 Met LysGln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe -25 -20 -15 AlaLeu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ser Leu Asn -10 -5 -1 1Asp Lys Pro Val Gly Phe Ala Ser Val Pro Thr Ala Asp Leu Pro Glu 5 10 15Gly Thr Val Gly Gly Leu Gly Gly Glu Ile Val Phe Val Arg Thr Ala 20 25 3035 Glu Glu Leu Glu Lys Tyr Thr Thr Ala Glu Gly Lys Tyr Val Ile Val 40 4550 Val Asp Gly Thr Ile Val Phe Glu Pro Lys Arg Glu Ile Lys Val Leu 55 6065 Ser Asp Lys Thr Ile Val Gly Ile Asn Asp Ala Lys Ile Val Gly Gly 70 7580 Gly Leu Val Ile Lys Asp Ala Gln Asn Val Ile Ile Arg Asn Ile His 85 9095 Phe Glu Gly Phe Tyr Met Glu Asp Asp Pro Arg Gly Lys Lys Tyr Asp 100105 110 115 Phe Asp Tyr Ile Asn Val Glu Asn Ser His His Ile Trp Ile AspHis 120 125 130 Cys Thr Phe Val Asn Gly Asn Asp Gly Ala Val Asp Ile LysLys Tyr 135 140 145 Ser Asn Tyr Ile Thr Val Ser Trp Cys Lys Phe Val AspHis Asp Lys 150 155 160 Val Ser Leu Val Gly Ser Ser Asp Lys Glu Asp ProGlu Gln Ala Gly 165 170 175 Gln Ala Tyr Lys Val Thr Tyr His His Asn TyrPhe Lys Asn Cys Ile 180 185 190 195 Gln Arg Met Pro Arg Ile Arg Phe GlyMet Ala His Val Phe Asn Asn 200 205 210 Phe Tyr Ser Met Gly Leu Arg ThrGly Val Ser Gly Asn Val Phe Pro 215 220 225 Ile Tyr Gly Val Ala Ser AlaMet Gly Ala Lys Val His Val Glu Gly 230 235 240 Asn Tyr Phe Met Gly TyrGly Ala Val Met Ala Glu Ala Gly Ile Ala 245 250 255 Phe Leu Pro Thr ArgIle Met Gly Pro Val Glu Gly Tyr Leu Thr Leu 260 265 270 275 Gly Glu GlyAsp Ala Lys Asn Glu Phe Tyr Tyr Cys Lys Glu Pro Glu 280 285 290 Val ArgPro Val Glu Glu Gly Lys Pro Ala Leu Asp Pro Arg Glu Tyr 295 300 305 TyrAsp Tyr Thr Leu Asp Pro Val Gln Asp Val Pro Lys Ile Val Val 310 315 320Asp Gly Ala Gly Ala Gly Lys Leu Val Phe Glu Glu Leu Asn Thr Ala 325 330335 Gln Arg Pro Pro Arg His Cys Gln Ser Gly Ile Leu Lys Arg Val 340 345350 4 1116 DNA Thermotoga maritima CDS (13)..(1113) 4 cagtagggag gg atgctc atg agg ttt tct cgt gtg gtt tct tta gta ctg 51 Met Leu Met Arg PheSer Arg Val Val Ser Leu Val Leu -25 -20 -15 ctt ctt gtt ttc aca gct gttcta act ggt gct gta aaa gct tct ctc 99 Leu Leu Val Phe Thr Ala Val LeuThr Gly Ala Val Lys Ala Ser Leu -10 -5 -1 1 aat gac aaa cct gtg gga tttgca tcc gta ccg acg gcg gat tta ccg 147 Asn Asp Lys Pro Val Gly Phe AlaSer Val Pro Thr Ala Asp Leu Pro 5 10 15 20 gag ggc aca gtt ggt gga ttgggt ggt gag atc gtt ttc gtc aga aca 195 Glu Gly Thr Val Gly Gly Leu GlyGly Glu Ile Val Phe Val Arg Thr 25 30 35 gcg gaa gaa ctg gag aaa tac acaaca gca gaa gga aag tac gta ata 243 Ala Glu Glu Leu Glu Lys Tyr Thr ThrAla Glu Gly Lys Tyr Val Ile 40 45 50 gtc gtt gat gga acg atc gtt ttt gaacca aag aga gaa att aaa gtt 291 Val Val Asp Gly Thr Ile Val Phe Glu ProLys Arg Glu Ile Lys Val 55 60 65 ctt tca gac aaa acg atc gtg gga ata aacgat gca aag ata gtc ggt 339 Leu Ser Asp Lys Thr Ile Val Gly Ile Asn AspAla Lys Ile Val Gly 70 75 80 gga ggt ctt gtg ata aag gat gcc cag aat gtgatc ata aga aat att 387 Gly Gly Leu Val Ile Lys Asp Ala Gln Asn Val IleIle Arg Asn Ile 85 90 95 100 cat ttt gag ggc ttt tac atg gag gac gat cctcgg ggt aag aag tat 435 His Phe Glu Gly Phe Tyr Met Glu Asp Asp Pro ArgGly Lys Lys Tyr 105 110 115 gat ttc gac tat atc aac gtg gaa aat tct catcat atc tgg atc gac 483 Asp Phe Asp Tyr Ile Asn Val Glu Asn Ser His HisIle Trp Ile Asp 120 125 130 cac tgt acc ttc gtc aac ggc aac gat ggt gcagtg gat att aaa aaa 531 His Cys Thr Phe Val Asn Gly Asn Asp Gly Ala ValAsp Ile Lys Lys 135 140 145 tac tca aac tac atc act gtt tcc tgg tgt aaattt gtg gat cac gac 579 Tyr Ser Asn Tyr Ile Thr Val Ser Trp Cys Lys PheVal Asp His Asp 150 155 160 aag gtc tcc ctc gtt ggt tcc tcc gac aaa gaagat ccg gaa cag gca 627 Lys Val Ser Leu Val Gly Ser Ser Asp Lys Glu AspPro Glu Gln Ala 165 170 175 180 ggg cag gct tac aag gtc acg tac cac cataac tac ttc aag aac tgt 675 Gly Gln Ala Tyr Lys Val Thr Tyr His His AsnTyr Phe Lys Asn Cys 185 190 195 att cag aga atg ccc aga att aga ttt ggaatg gca cac gtg ttc aat 723 Ile Gln Arg Met Pro Arg Ile Arg Phe Gly MetAla His Val Phe Asn 200 205 210 aac ttc tac agc atg ggc ctg aga aca ggtgtc tct gga aac gtc ttc 771 Asn Phe Tyr Ser Met Gly Leu Arg Thr Gly ValSer Gly Asn Val Phe 215 220 225 ccc att tac ggt gtt gct tca gcg atg ggagcg aaa gtc cac gtt gaa 819 Pro Ile Tyr Gly Val Ala Ser Ala Met Gly AlaLys Val His Val Glu 230 235 240 gga aac tac ttc atg gga tac ggt gct gtgatg gca gag gcg gga att 867 Gly Asn Tyr Phe Met Gly Tyr Gly Ala Val MetAla Glu Ala Gly Ile 245 250 255 260 gcg ttc ctt ccc acc aga atc atg ggtccc gtg gaa ggt tat ctg acg 915 Ala Phe Leu Pro Thr Arg Ile Met Gly ProVal Glu Gly Tyr Leu Thr 265 270 275 ctc ggt gaa gga gat gca aag aat gaattt tac tac tgt aaa gaa cct 963 Leu Gly Glu Gly Asp Ala Lys Asn Glu PheTyr Tyr Cys Lys Glu Pro 280 285 290 gaa gtg cgt cct gtt gag gaa gga aaaccc gct ctc gat cca cgc gag 1011 Glu Val Arg Pro Val Glu Glu Gly Lys ProAla Leu Asp Pro Arg Glu 295 300 305 tac tac gat tac acg ctt gat cca gttcaa gat gtt cca aaa atc gtt 1059 Tyr Tyr Asp Tyr Thr Leu Asp Pro Val GlnAsp Val Pro Lys Ile Val 310 315 320 gta gat gga gca gga gca ggg aaa ctggtg ttt gaa gaa cta aat acg 1107 Val Asp Gly Ala Gly Ala Gly Lys Leu ValPhe Glu Glu Leu Asn Thr 325 330 335 340 gct cag taa 1116 Ala Gln 5 367PRT Thermotoga maritima 5 Met Leu Met Arg Phe Ser Arg Val Val Ser LeuVal Leu Leu Leu Val -25 -20 -15 -10 Phe Thr Ala Val Leu Thr Gly Ala ValLys Ala Ser Leu Asn Asp Lys -5 -1 1 5 Pro Val Gly Phe Ala Ser Val ProThr Ala Asp Leu Pro Glu Gly Thr 10 15 20 Val Gly Gly Leu Gly Gly Glu IleVal Phe Val Arg Thr Ala Glu Glu 25 30 35 Leu Glu Lys Tyr Thr Thr Ala GluGly Lys Tyr Val Ile Val Val Asp 40 45 50 55 Gly Thr Ile Val Phe Glu ProLys Arg Glu Ile Lys Val Leu Ser Asp 60 65 70 Lys Thr Ile Val Gly Ile AsnAsp Ala Lys Ile Val Gly Gly Gly Leu 75 80 85 Val Ile Lys Asp Ala Gln AsnVal Ile Ile Arg Asn Ile His Phe Glu 90 95 100 Gly Phe Tyr Met Glu AspAsp Pro Arg Gly Lys Lys Tyr Asp Phe Asp 105 110 115 Tyr Ile Asn Val GluAsn Ser His His Ile Trp Ile Asp His Cys Thr 120 125 130 135 Phe Val AsnGly Asn Asp Gly Ala Val Asp Ile Lys Lys Tyr Ser Asn 140 145 150 Tyr IleThr Val Ser Trp Cys Lys Phe Val Asp His Asp Lys Val Ser 155 160 165 LeuVal Gly Ser Ser Asp Lys Glu Asp Pro Glu Gln Ala Gly Gln Ala 170 175 180Tyr Lys Val Thr Tyr His His Asn Tyr Phe Lys Asn Cys Ile Gln Arg 185 190195 Met Pro Arg Ile Arg Phe Gly Met Ala His Val Phe Asn Asn Phe Tyr 200205 210 215 Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn Val Phe Pro IleTyr 220 225 230 Gly Val Ala Ser Ala Met Gly Ala Lys Val His Val Glu GlyAsn Tyr 235 240 245 Phe Met Gly Tyr Gly Ala Val Met Ala Glu Ala Gly IleAla Phe Leu 250 255 260 Pro Thr Arg Ile Met Gly Pro Val Glu Gly Tyr LeuThr Leu Gly Glu 265 270 275 Gly Asp Ala Lys Asn Glu Phe Tyr Tyr Cys LysGlu Pro Glu Val Arg 280 285 290 295 Pro Val Glu Glu Gly Lys Pro Ala LeuAsp Pro Arg Glu Tyr Tyr Asp 300 305 310 Tyr Thr Leu Asp Pro Val Gln AspVal Pro Lys Ile Val Val Asp Gly 315 320 325 Ala Gly Ala Gly Lys Leu ValPhe Glu Glu Leu Asn Thr Ala Gln 330 335 340 6 1113 DNA ArtificialSequence Synthetic 6 atg aaa caa caa aaa cgg ctt tac gcc cga ttg ctg acgctg tta ttt 48 Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr LeuLeu Phe -25 -20 -15 gcg ctc atc ttc ttg ctg cct cat tct gca gcc gcg gcatct ctc aat 96 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala SerLeu Asn -10 -5 -1 1 gac aaa cct gtg gga ttt gca tcc gta ccg acg gcg gattta ccg gag 144 Asp Lys Pro Val Gly Phe Ala Ser Val Pro Thr Ala Asp LeuPro Glu 5 10 15 ggc aca gtt ggt gga ttg ggt ggt gag atc gtt ttc gtc agaaca gcg 192 Gly Thr Val Gly Gly Leu Gly Gly Glu Ile Val Phe Val Arg ThrAla 20 25 30 35 gaa gaa ctg gag aaa tac aca acg gca gaa gga aag tac gtaata gtc 240 Glu Glu Leu Glu Lys Tyr Thr Thr Ala Glu Gly Lys Tyr Val IleVal 40 45 50 gtt gat gga acg atc gtt ttt gaa cca aag aga gaa att aaa gttctt 288 Val Asp Gly Thr Ile Val Phe Glu Pro Lys Arg Glu Ile Lys Val Leu55 60 65 tca gac aaa acg atc gtg gga ata aac gat gca aag ata gtc ggt gga336 Ser Asp Lys Thr Ile Val Gly Ile Asn Asp Ala Lys Ile Val Gly Gly 7075 80 ggt ctt gtg ata aag gat gcc cag aat gtg atc ata aga aat att cat384 Gly Leu Val Ile Lys Asp Ala Gln Asn Val Ile Ile Arg Asn Ile His 8590 95 ttt gag ggc ttt tac atg gag gac gat cct cgg ggt aag aag tat gat432 Phe Glu Gly Phe Tyr Met Glu Asp Asp Pro Arg Gly Lys Lys Tyr Asp 100105 110 115 ttc gac tat atc aac gtg gaa aat tct cat cat atc tgg atc gaccac 480 Phe Asp Tyr Ile Asn Val Glu Asn Ser His His Ile Trp Ile Asp His120 125 130 tgt acc ttc gtc aac ggc aac gat ggt gca gtg gat att aaa aaatac 528 Cys Thr Phe Val Asn Gly Asn Asp Gly Ala Val Asp Ile Lys Lys Tyr135 140 145 tca aac tac atc act gtt tcc tgg tgt aaa ttt gtg gat cac gacaag 576 Ser Asn Tyr Ile Thr Val Ser Trp Cys Lys Phe Val Asp His Asp Lys150 155 160 gtc tcc ctc gtt ggt tcc tcc gac aaa gaa gat ccg gaa cag gcaggg 624 Val Ser Leu Val Gly Ser Ser Asp Lys Glu Asp Pro Glu Gln Ala Gly165 170 175 cag gct tac aag gtc acg tac cac cat aac tac ttc aag aac tgtatt 672 Gln Ala Tyr Lys Val Thr Tyr His His Asn Tyr Phe Lys Asn Cys Ile180 185 190 195 cag aga atg ccc aga att aga ttt gga atg gca cac gtg ttcaat aac 720 Gln Arg Met Pro Arg Ile Arg Phe Gly Met Ala His Val Phe AsnAsn 200 205 210 ttc tac agc atg ggc ctg aga aca ggt gtc tct gga aac gtcttc ccc 768 Phe Tyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn Val PhePro 215 220 225 att tac ggt gtt gct tca gcg atg gga gcg aaa gtc cac gttgaa gga 816 Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val His Val GluGly 230 235 240 aac tac ttc atg gga tac ggt gct gtg atg gca gag gcg ggaatt gcg 864 Asn Tyr Phe Met Gly Tyr Gly Ala Val Met Ala Glu Ala Gly IleAla 245 250 255 ttc ctt ccc acc aga atc atg ggt ccc gtg gaa ggt tat ctgacg ctc 912 Phe Leu Pro Thr Arg Ile Met Gly Pro Val Glu Gly Tyr Leu ThrLeu 260 265 270 275 ggt gaa gga gat gca aag aat gaa ttt tac tac tgt aaagaa cct gaa 960 Gly Glu Gly Asp Ala Lys Asn Glu Phe Tyr Tyr Cys Lys GluPro Glu 280 285 290 gtg cgt cct gtt gag gaa gga aaa ccc gct ctc gat ccacgc gag tac 1008 Val Arg Pro Val Glu Glu Gly Lys Pro Ala Leu Asp Pro ArgGlu Tyr 295 300 305 tac gat tac acg ctt gat cca gtt caa gat gtt cca aaaatc gtt gta 1056 Tyr Asp Tyr Thr Leu Asp Pro Val Gln Asp Val Pro Lys IleVal Val 310 315 320 gat gga gca gga gca ggg aaa ctg gtg ttt gaa gaa ctaaat acg gct 1104 Asp Gly Ala Gly Ala Gly Lys Leu Val Phe Glu Glu Leu AsnThr Ala 325 330 335 cag tgataa 1113 Gln 340 7 369 PRT ArtificialSequence Synthetic 7 Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu ThrLeu Leu Phe -25 -20 -15 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala AlaAla Ser Leu Asn -10 -5 -1 1 Asp Lys Pro Val Gly Phe Ala Ser Val Pro ThrAla Asp Leu Pro Glu 5 10 15 Gly Thr Val Gly Gly Leu Gly Gly Glu Ile ValPhe Val Arg Thr Ala 20 25 30 35 Glu Glu Leu Glu Lys Tyr Thr Thr Ala GluGly Lys Tyr Val Ile Val 40 45 50 Val Asp Gly Thr Ile Val Phe Glu Pro LysArg Glu Ile Lys Val Leu 55 60 65 Ser Asp Lys Thr Ile Val Gly Ile Asn AspAla Lys Ile Val Gly Gly 70 75 80 Gly Leu Val Ile Lys Asp Ala Gln Asn ValIle Ile Arg Asn Ile His 85 90 95 Phe Glu Gly Phe Tyr Met Glu Asp Asp ProArg Gly Lys Lys Tyr Asp 100 105 110 115 Phe Asp Tyr Ile Asn Val Glu AsnSer His His Ile Trp Ile Asp His 120 125 130 Cys Thr Phe Val Asn Gly AsnAsp Gly Ala Val Asp Ile Lys Lys Tyr 135 140 145 Ser Asn Tyr Ile Thr ValSer Trp Cys Lys Phe Val Asp His Asp Lys 150 155 160 Val Ser Leu Val GlySer Ser Asp Lys Glu Asp Pro Glu Gln Ala Gly 165 170 175 Gln Ala Tyr LysVal Thr Tyr His His Asn Tyr Phe Lys Asn Cys Ile 180 185 190 195 Gln ArgMet Pro Arg Ile Arg Phe Gly Met Ala His Val Phe Asn Asn 200 205 210 PheTyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn Val Phe Pro 215 220 225Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val His Val Glu Gly 230 235240 Asn Tyr Phe Met Gly Tyr Gly Ala Val Met Ala Glu Ala Gly Ile Ala 245250 255 Phe Leu Pro Thr Arg Ile Met Gly Pro Val Glu Gly Tyr Leu Thr Leu260 265 270 275 Gly Glu Gly Asp Ala Lys Asn Glu Phe Tyr Tyr Cys Lys GluPro Glu 280 285 290 Val Arg Pro Val Glu Glu Gly Lys Pro Ala Leu Asp ProArg Glu Tyr 295 300 305 Tyr Asp Tyr Thr Leu Asp Pro Val Gln Asp Val ProLys Ile Val Val 310 315 320 Asp Gly Ala Gly Ala Gly Lys Leu Val Phe GluGlu Leu Asn Thr Ala 325 330 335 Gln 340 8 1113 DNA Artificial SequenceSynthetic 8 atg aaa caa caa aaa cgg ctt tac gcc cga ttg ctg acg ctg ttattt 48 Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe-25 -20 -15 gcg ctc atc ttc ttg ctg cct cat tct gca gcc gcg gca tct ctcaat 96 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ser Leu Asn-10 -5 -1 1 gac aaa cct gtg gga ttt gca tcc gta ccg acg gcg gat tta ccggag 144 Asp Lys Pro Val Gly Phe Ala Ser Val Pro Thr Ala Asp Leu Pro Glu5 10 15 ggc aca gtt ggt gga ttg ggt ggt gag atc gtt ttc gtc aga aca gcg192 Gly Thr Val Gly Gly Leu Gly Gly Glu Ile Val Phe Val Arg Thr Ala 2025 30 35 gaa gaa ctg gag aaa tac aca acg gca gaa gga aag tac gta ata gtc240 Glu Glu Leu Glu Lys Tyr Thr Thr Ala Glu Gly Lys Tyr Val Ile Val 4045 50 gtt gat gga acg atc gtt ttt gaa cca aag aga gaa att aaa gtt ctt288 Val Asp Gly Thr Ile Val Phe Glu Pro Lys Arg Glu Ile Lys Val Leu 5560 65 tca gac aaa acg atc gtg gga ata aac gat gca aag ata gtc ggt gga336 Ser Asp Lys Thr Ile Val Gly Ile Asn Asp Ala Lys Ile Val Gly Gly 7075 80 ggt ctt gtg ata aag gat gcc cag aat gtg atc ata aga aat att cat384 Gly Leu Val Ile Lys Asp Ala Gln Asn Val Ile Ile Arg Asn Ile His 8590 95 ttt gag ggc ttt tac atg gag gac gat cct cgg ggt aag aag tat gat432 Phe Glu Gly Phe Tyr Met Glu Asp Asp Pro Arg Gly Lys Lys Tyr Asp 100105 110 115 ttc gac tat atc aac gtg gaa aat tct cat cat atc tgg atc gaccac 480 Phe Asp Tyr Ile Asn Val Glu Asn Ser His His Ile Trp Ile Asp His120 125 130 att acc ttc gtc aac ggc aac gat ggt gca gtg gat att aaa aaatac 528 Ile Thr Phe Val Asn Gly Asn Asp Gly Ala Val Asp Ile Lys Lys Tyr135 140 145 tca aac tac atc act gtt tcc tgg aac aaa ttt gtg gat cac gacaag 576 Ser Asn Tyr Ile Thr Val Ser Trp Asn Lys Phe Val Asp His Asp Lys150 155 160 gtc tcc ctc gtt ggt tcc tcc gac aaa gaa gat ccg gaa cag gcaggg 624 Val Ser Leu Val Gly Ser Ser Asp Lys Glu Asp Pro Glu Gln Ala Gly165 170 175 cag gct tac aag gtc acg tac cac cat aac tac ttc aag aac ctgatt 672 Gln Ala Tyr Lys Val Thr Tyr His His Asn Tyr Phe Lys Asn Leu Ile180 185 190 195 cag aga atg ccc aga att aga ttt gga atg gca cac gtg ttcaat aac 720 Gln Arg Met Pro Arg Ile Arg Phe Gly Met Ala His Val Phe AsnAsn 200 205 210 ttc tac agc atg ggc ctg aga aca ggt gtc tct gga aac gtcttc ccc 768 Phe Tyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn Val PhePro 215 220 225 att tac ggt gtt gct tca gcg atg gga gcg aaa gtc cac gttgaa gga 816 Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val His Val GluGly 230 235 240 aac tac ttc atg gga tac ggt gct gtg atg gca gag gcg ggaatt gcg 864 Asn Tyr Phe Met Gly Tyr Gly Ala Val Met Ala Glu Ala Gly IleAla 245 250 255 ttc ctt ccc acc aga atc atg ggt ccc gtg gaa ggt tat ctgacg ctc 912 Phe Leu Pro Thr Arg Ile Met Gly Pro Val Glu Gly Tyr Leu ThrLeu 260 265 270 275 ggt gaa gga gat gca aag aat gaa ttt tac tac tgt aaagaa cct gaa 960 Gly Glu Gly Asp Ala Lys Asn Glu Phe Tyr Tyr Cys Lys GluPro Glu 280 285 290 gtg cgt cct gtt gag gaa gga aaa ccc gct ctc gat ccacgc gag tac 1008 Val Arg Pro Val Glu Glu Gly Lys Pro Ala Leu Asp Pro ArgGlu Tyr 295 300 305 tac gat tac acg ctt gat cca gtt caa gat gtt cca aaaatc gtt gta 1056 Tyr Asp Tyr Thr Leu Asp Pro Val Gln Asp Val Pro Lys IleVal Val 310 315 320 gat gga gca gga gca ggg aaa ctg gtg ttt gaa gaa ctaaat acg gct 1104 Asp Gly Ala Gly Ala Gly Lys Leu Val Phe Glu Glu Leu AsnThr Ala 325 330 335 cag tgataa 1113 Gln 340 9 369 PRT ArtificialSequence Synthetic 9 Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu ThrLeu Leu Phe -25 -20 -15 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala AlaAla Ser Leu Asn -10 -5 -1 1 Asp Lys Pro Val Gly Phe Ala Ser Val Pro ThrAla Asp Leu Pro Glu 5 10 15 Gly Thr Val Gly Gly Leu Gly Gly Glu Ile ValPhe Val Arg Thr Ala 20 25 30 35 Glu Glu Leu Glu Lys Tyr Thr Thr Ala GluGly Lys Tyr Val Ile Val 40 45 50 Val Asp Gly Thr Ile Val Phe Glu Pro LysArg Glu Ile Lys Val Leu 55 60 65 Ser Asp Lys Thr Ile Val Gly Ile Asn AspAla Lys Ile Val Gly Gly 70 75 80 Gly Leu Val Ile Lys Asp Ala Gln Asn ValIle Ile Arg Asn Ile His 85 90 95 Phe Glu Gly Phe Tyr Met Glu Asp Asp ProArg Gly Lys Lys Tyr Asp 100 105 110 115 Phe Asp Tyr Ile Asn Val Glu AsnSer His His Ile Trp Ile Asp His 120 125 130 Ile Thr Phe Val Asn Gly AsnAsp Gly Ala Val Asp Ile Lys Lys Tyr 135 140 145 Ser Asn Tyr Ile Thr ValSer Trp Asn Lys Phe Val Asp His Asp Lys 150 155 160 Val Ser Leu Val GlySer Ser Asp Lys Glu Asp Pro Glu Gln Ala Gly 165 170 175 Gln Ala Tyr LysVal Thr Tyr His His Asn Tyr Phe Lys Asn Leu Ile 180 185 190 195 Gln ArgMet Pro Arg Ile Arg Phe Gly Met Ala His Val Phe Asn Asn 200 205 210 PheTyr Ser Met Gly Leu Arg Thr Gly Val Ser Gly Asn Val Phe Pro 215 220 225Ile Tyr Gly Val Ala Ser Ala Met Gly Ala Lys Val His Val Glu Gly 230 235240 Asn Tyr Phe Met Gly Tyr Gly Ala Val Met Ala Glu Ala Gly Ile Ala 245250 255 Phe Leu Pro Thr Arg Ile Met Gly Pro Val Glu Gly Tyr Leu Thr Leu260 265 270 275 Gly Glu Gly Asp Ala Lys Asn Glu Phe Tyr Tyr Cys Lys GluPro Glu 280 285 290 Val Arg Pro Val Glu Glu Gly Lys Pro Ala Leu Asp ProArg Glu Tyr 295 300 305 Tyr Asp Tyr Thr Leu Asp Pro Val Gln Asp Val ProLys Ile Val Val 310 315 320 Asp Gly Ala Gly Ala Gly Lys Leu Val Phe GluGlu Leu Asn Thr Ala 325 330 335 Gln 340 10 42 DNA Artificial SequencePrimer 10 gtcgccgggg cggccgctat caattggtaa ctgtatctca gc 42 11 64 DNAArtificial Sequence Primer 11 gtcgcccggg agctctgatc aggtaccaagcttgtcgacc tgcagaatga ggcagcaaga 60 agat 64 12 61 DNA ArtificialSequence Primer 12 gtcggcggcc gctgatcacg taccaagctt gtcgacctgcagaatgaggc agcaagaaga 60 t 61 13 35 DNA Artificial Sequence Primer 13gtcggagctc tatcaattgg taactgtatc tcagc 35 14 35 DNA Artificial SequencePrimer 14 aacagctgat cacgactgat cttttagctt ggcac 35 15 37 DNA ArtificialSequence Primer 15 aactgcagcc gcggcacatc ataatgggac aaatggg 37 16 34 DNAArtificial Sequence Primer 16 aaaccgcggc atctctcaat gacaaacctg tggg 3417 38 DNA Artificial Sequence Primer 17 aaagcggccg ctgagccgta tttagttcttcaaacacc 38 18 29 DNA Artificial Sequence Primer 18 gggaattcttactgagccgt atttagttc 29 19 28 DNA Artificial Sequence Primer 19ccggatccag tagggaggga tgctcatg 28 20 41 DNA Artificial Sequence Primer20 cattctgcag ccgcggcatc tctcaatgac aaacctgtgg g 41 21 51 DNA ArtificialSequence Primer 21 catcatggat ccgcggccgc ttatcactga gccgtatttagttcttcaaa c 51 22 41 DNA Artificial Sequence Primer 22 cattctgcagccgcggcatc tctcaatgac aaacctgtgg g 41 23 117 DNA Artificial SequencePrimer 23 ccaacgaggg agaccttgtc gtgatccaca aatttgytcc aggaaacagtgatgtagttt 60 gagtattttt taatatccac tgcaccatcg ttgccgttga cgaaggtawygtggtcg 117 24 114 DNA Artificial Sequence Primer 24 ggatcacgacaaggtctccc tcgttggttc ctccgacaaa gaagatccgg aacaggcagg 60 gcaggcttacaaggtcacgt accaccataa ctacttcaag aacctgattc agag 114 25 51 DNAArtificial Sequence Primer 25 catcatggat ccgcggccgc ttatcactgagccgtattta gttcttcaaa c 51

1. A bacterial host cell transformed with a vector comprising a DNAsequence that is endogenous to a strain of Thermotoga maritima or avariant of the DNA sequence, which DNA sequence or variant DNA sequenceencodes for a pectate lyase polypeptide (EC 4.2.2.2).
 2. The host cellof claim 1, wherein the strain of Thermotoga maritima is the strainThermotoga maritima, DSM
 3109. 3. The host cell of claim 1 which isneutralophilic, alkalophilic, mesophilic or thermophilic.
 4. The hostcell of claim 1 which is a Bacillus host cell.
 5. The host cell of claim4, which is a Bacillus amyloliquefaciens, Bacillus licheniformis,Bacillus megaterium, Bacillus stearothermophilus or Bacillus subtilisrcell.
 6. The host cell of claim 1, wherein the vector is integrated intothe genome of the host.
 7. The host cell of claim 1, wherein the vectoris integrated into the genome of the untransformed host.
 8. The hostcell of claim 1, wherein the vector is present as an expression plasmid.9. The host cell of claim 8, wherein the vector has been amplified onthe genome or the expression plasmid is a multi-copy plasmid.
 10. Abacterial expression vector which carries an inserted DNA sequenceencoding for a pectate lyase polypeptide (EC 4.2.2.2) endogenous to astrain of Thermotoga maritima or a variant of the pectate lyasepolypeptide.
 11. The vector of claim 10 in which the expression cassettecomprises regulatory regions from a species of Bacillus.
 12. The vectorof claim 11, wherein the Bacillus sp. regulatory regions are endogeneousto the host.
 13. A method for producing a pectate lyase (EC 4.2.2.2)polypeptide endogenous to a strain of Thermotoga maritima or a variantof the pectate lyase polypeptide, the method comprising the steps of:(a) growing a bacterial host cell in a nutrient medium, under conditionsto overproduce the pectate lyase polypeptide, wherein the bacterial hostcell has been en transformed with an expression cassette which includes,as operably joined components, (i) a transcriptional and translationalinitiation regulatory region, (ii) a DNA sequence encoding the pectatelyase polypeptide, (iii) a transcriptional and translational terminationregulatory region, wherein the regulatory regions are functional in thehost, and (iv) a selection marker gene for selecting transformed hostcells; and (b) recovering the pectate lyase polypeptide.
 14. Apolypeptide having pectate lyase activity (EC 4.2.2.2), whichpolypeptide is selected from the group consisting of (a) polypeptideshaving pectate lyases activity, wherein the polypeptide is encoded by aDNA sequence endogenous to a strain of Thermotoga maritima; and (b) sitedirected variants of the polypeptide encoded by a DNA sequenceendogenous to a strain of Thermotoga maritima, wherein one, two, threeor four cysteine residues have been altered to other amino acidresidues.
 15. The polypeptide of claim 14, wherein three cysteineresidues have been altered to other amino acid residues.
 16. Thepolypeptide of claim 15, wherein the cysteine residues independently ofeach other have been altered to asparagines, isoleucine or leucine. 17.The polypeptide of claim 14, wherein the strain of Thermotoga maritimais the strain Thermotoga maritima, DSM
 3109. 18. The polypeptide ofclaim 16, which variant has amino acid substitutions in positions 161,185 and 223 relative to the amino acid numbering of SEQ ID NO:
 3. 19.The polypeptide of claim 16, which variant has a catalytically activedomain represented by positions 30 to 369 of SEQ ID NO:
 9. 20. A methodfor optimizing pectate lyase expression in a bacterial host, the methodcomprising the steps of: (a) in the host, expressing a pectate lyasepolypeptide fused to a reporter molecule; (b) in the supernatant of thefermented host, monitoring the concentration of expressed pectate lyasepolypeptide by measuring the intrinsic property or properties of thereporter molecule.
 21. The method of claim 20, wherein the reportermolecule is a Green Fluorescent Protein, and the intrinsic property isfluorescence emission.
 22. A polypeptide hybrid consisting essentiallyof a pectate lyase polypeptide fused to a green fluorescent protein. 23.A method of producing the hybrid of claim 22, comprising (a) growing atransformed host under conditions to express the hybrid whereby thetransformed culture is substantially free of untransformed cells; (b)incubating the transformed culture in a nutrient medium, whereby thehybrid is overproduced; and (c) recovering the hybrid.
 24. A detergentcomposition comprising the polypeptide of claim 14 and a surfactant. 25.A process for machine treatment of a fabric, comprising treating thefabric during a washing cycle of a machine washing process with awashing solution containing the polypeptide of claim 14 and asurfactant.
 26. The process of claim 25, wherein the fabric is made offibers selected from the group consisting of hemp, jute, flax and linen.27. The process of claim 26, wherein the washing solution is addedduring a textile scouring process step.